A neurotoxic peripherin splice variant in a mouse model of ALS.

Peripherin, a neuronal intermediate filament (nIF) protein found associated with pathological aggregates in motor neurons of patients with amyotrophic lateral sclerosis (ALS) and of transgenic mice overexpressing mutant superoxide dismutase-1 (SOD1G37R), induces the selective degeneration of motor neurons when overexpressed in transgenic mice. Mouse peripherin is unique compared with other nIF proteins in that three peripherin isoforms are generated by alternative splicing. Here, the properties of the peripherin splice variants Per 58, Per 56, and Per 61 have been investigated in transfected cell lines, in primary motor neurons, and in transgenic mice overexpressing peripherin or overexpressing SOD1G37R. Of the three isoforms, Per 61 proved to be distinctly neurotoxic, being assembly incompetent and inducing degeneration of motor neurons in culture. Using isoform-specific antibodies, Per 61 expression was detected in motor neurons of SOD1G37R transgenic mice but not of control or peripherin transgenic mice. The Per 61 antibody also selectively labeled motor neurons and axonal spheroids in two cases of familial ALS and immunoprecipitated a higher molecular mass peripherin species from disease tissue. This evidence suggests that expression of neurotoxic splice variants of peripherin may contribute to the neurodegenerative mechanism in ALS.

High threshold for induction of the stress response in motor neurons is associated with failure to activate HSF1.

Heat shock protein 70 (Hsp70) protects cultured motor neurons from the toxic effects of mutations in Cu/Zn-superoxide dismutase (SOD-1), which is responsible for a familial form of the disease, amyotrophic lateral sclerosis (ALS). Here, the endogenous heat shock response of motor neurons was investigated to determine whether a high threshold for activating this protective mechanism contributes to their vulnerability to stresses associated with ALS. When heat shocked, cultured motor neurons failed to express Hsp70 or transactivate a green fluorescent protein reporter gene driven by the Hsp70 promoter, although Hsp70 was induced in glial cells. No increase in Hsp70 occurred in motor neurons after exposure to excitotoxic glutamate or expression of mutant SOD-1 with a glycine--> alanine substitution at residue 93 (G93A), nor was Hsp70 increased in spinal cords of G93A SOD-1 transgenic mice or sporadic or familial ALS patients. In contrast, strong Hsp70 induction occurred in motor neurons with expression of a constitutively active form of heat shock transcription factor (HSF)-1 or when proteasome activity was sufficiently inhibited to induce accumulation of an alternative transcription factor HSF2. These results indicate that the high threshold for induction of the stress response in motor neurons stems from an impaired ability to activate the main heat shock-stress sensor, HSF1.

Induction of multiple heat shock proteins and neuroprotection in a primary culture model of familial amyotrophic lateral sclerosis.

High threshold for stress-induced activation of the heat shock transcription factor, Hsf1, may contribute to vulnerability of motor neurons to disease and limit efficacy of agents promoting expression of neuroprotective heat shock proteins (Hsps) through this transcription factor. Plasmid encoding a constitutively active form of Hsf1, Hsf1act, and chemicals shown to activate Hsf1 in other cells were investigated in a primary culture model of familial amyotrophic lateral sclerosis. Hsf1act and the Hsp90 inhibitor, geldanamycin, induced high expression of multiple Hsps in cultured motor neurons and conferred dramatic neuroprotection against SOD1G93A in comparison to Hsp70 or Hsp25 alone. Two other Hsp90 inhibitors, 17-allylamino-17-demethoxygeldanamycin (17-AAG) and radicicol, and pyrrolidine dithiocarbamate induced robust expression of Hsp70 and Hsp40 in motor neurons, but at cytotoxic concentrations. 17-AAG, which penetrates the blood-brain barrier, has exhibited a higher therapeutic index than geldanamycin, but this may not be the case when activation of Hsf1 in neurons is targeted.

Adeno-associated virus-mediated gene transfer of human aromatic L-amino acid decarboxylase protects mixed striatal primary cultures from L-DOPA toxicity.

Although L-DOPA is the drug of choice for Parkinson's disease, prolonged L-DOPA therapy results in decreased drug effectiveness and the appearance of motor complications. This may be due in part to the progressive loss of the enzyme, aromatic L-amino acid decarboxylase (AADC). We have developed an adeno-associated virus vector (AAV-hAADC) that contains human AADC cDNA under the control of the cytomegalovirus promoter. Infusion of this vector into the striatum of parkinsonian rats and monkeys improves L-DOPA responsiveness by improving AADC-mediated conversion of L-DOPA to dopamine. This is now the basis of a proposed therapy for advanced Parkinson's disease. A key concern has been that over-production of dopamine in striatal neurons could cause dopamine toxicity. To investigate this possibility in a controlled system, mixed striatal primary rat neuronal cultures were prepared. Exposure of cultures to high concentrations of L-DOPA induced the following changes: cell death in nigral and striatal neurons, aggregation of neurofilaments and focal axonal swellings, abnormal expression of DARPP-32, and activation of astroglia and microglial cells. Transduction of cultures with AAV-hAADC resulted in efficient and sustained neuronal expression of the AADC protein and prevented all the L-DOPA-induced toxicities. The protective effects were due primarily to AADC-dependent conversion of L-DOPA to dopamine and an increase in induction of vesicular monoamine transporter resulting in dopamine storage in cultured cells. These results suggest a neuroprotective role for AADC gene transfer against L-DOPA toxicity.

Novel caprine adeno-associated virus (AAV) capsid (AAV-Go.1) is closely related to the primate AAV-5 and has unique tropism and neutralization properties.

Preexisting humoral immunity to adeno-associated virus (AAV) vectors may limit their clinical utility in gene delivery. We describe a novel caprine AAV (AAV-Go.1) capsid with unique biological properties. AAV-Go.1 capsid was cloned from goat-derived adenovirus preparations. Surprisingly, AAV-Go.1 capsid was 94% identical to the human AAV-5, with differences predicted to be largely on the surface and on or under the spike-like protrusions. In an in vitro neutralization assay using human immunoglobulin G (IgG) (intravenous immune globulin [IVIG]), AAV-Go.1 had higher resistance than AAV-5 (100-fold) and resistance similar to that of AAV-4 or AAV-8. In an in vivo model, SCID mice were pretreated with IVIG to generate normal human IgG plasma levels prior to the administration of AAV human factor IX vectors. Protein expression after intramuscular administration of AAV-Go.1 was unaffected in IVIG-pretreated mice, while it was reduced 5- and 10-fold after administration of AAV-1 and AAV-8, respectively. In contrast, protein expression after intravenous administration of AAV-Go.1 was reduced 7.1-fold, similar to the 3.8-fold reduction observed after AAV-8 administration in IVIG-pretreated mice, and protein expression was essentially extinguished after AAV-2 administration in mice pretreated with much less IVIG (15-fold). AAV-Go.1 vectors also demonstrated a marked tropism for lung when administered intravenously in SCID mice. The pulmonary tropism and high neutralization resistance to human preexisting antibodies suggest novel therapeutic uses for AAV-Go.1 vectors, including targeting diseases such as cystic fibrosis. Nonprimate sources of AAVs may be useful to identify additional capsids with distinct tropisms and high resistance to neutralization by human preexisting antibodies.

Expression of a membrane-bound form of the ferroxidase ceruloplasmin by leptomeningeal cells.

Ceruloplasmin is a key enzyme involved in detoxifying ferrous iron, which can generate free radicals. The secreted form of ceruloplasmin is produced by the liver and is abundant in serum. We have previously identified a membrane-bound glycosylphosphatidylinositol (GPI)-anchored form of ceruloplasmin (GPI-Cp) that is expressed by astrocytes in the central nervous system (CNS) (Patel and David. 1997. J Biol Chem 272:20185-20190). We now provide direct evidence that rat leptomeningeal cells, which cover the surface of the brain, also express GPI-Cp. The expression of GPI-Cp on the surface of these cells increases with postnatal development and is regulated in vitro by cell density, time in culture, and various extracellular matrix molecules. The expression of GPI-Cp also appears to be regulated differently in astrocytes and leptomeningeal cells in vitro. The abundant expression of GPI-Cp on the surface of leptomeningeal cells suggests that these cells play a role in antioxidant defense along the surface of the postnatal CNS possibly by detoxifying the cerebrospinal fluid.

Striatal delivery of rAAV-hAADC to rats with preexisting immunity to AAV.

We tested the hypotheses that initial immunization of rats with rAAV might limit subsequent transduction by rAAV-hAADC when stereotaxically infused into the striatum and that the level of inhibition would correlate with AAV neutralizing antibody titers. Immunohistochemical detection of AADC and analysis by stereology revealed that the control group (no immunization) had the greatest volume of distribution of AADC (20.32 +/- 2.03 mm3) (+/-SD). There was a 58% decrease in spread (8.46 +/- 3.67 mm3, P < 0.008) in the high-dose immunization group (5 x 10(10) vg rAAV-null). Transduction weakly correlated with preexisting titer levels of neutralizing antibody at the time of intrastriatal rAAV-hAADC infusion. Only rats with neutralizing antibody titers of 1:1208 +/- 332 had significantly decreased AADC transgene expression compared to the unimmunized control group. Immunohistochemistry on serial sections for inflammatory markers including GFAP, CD11b, CD4, and CD8a revealed normal morphology and no cellular infiltration, suggesting little immune reaction in the CNS. We conclude that rAAV vectors can transduce brain tissue in the context of preexisting immunity, but that efficiency of transduction declines significantly in the presence of very high titers of neutralizing antibodies. These results have important implications for gene therapy for CNS disorders.