The Aggregation Inhibitor Peptide QBP1 as a Therapeutic Molecule for the Polyglutamine Neurodegenerative Diseases

Misfolding and abnormal aggregation of proteins in the brain are implicated in the pathogenesis of various neurodegenerative diseases including Alzheimer's, Parkinson's, and the polyglutamine (polyQ) diseases. In the polyQ diseases, an abnormally expanded polyQ stretch triggers misfolding and aggregation of the disease-causing proteins, eventually resulting in neurodegeneration. In this paper, we introduce our therapeutic strategy against the polyQ diseases using polyQ binding peptide 1 (QBP1), a peptide that we identified by phage display screening. We showed that QBP1 specifically binds to the expanded polyQ stretch and inhibits its misfolding and aggregation, resulting in suppression of neurodegeneration in cell culture and animal models of the polyQ diseases. We further demonstrated the potential of protein transduction domains (PTDs) for in vivo delivery of QBP1. We hope that in the near future, chemical analogues of aggregation inhibitor peptides including QBP1 will be developed against protein misfolding-associated neurodegenerative diseases

Transgenic Monkey Model of the Polyglutamine Diseases Recapitulating Progressive Neurological Symptoms

Age-associated neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and the polyglutamine (polyQ) diseases, are becoming prevalent as a consequence of elongation of the human lifespan. Although various rodent models have been developed to study and overcome these diseases, they have limitations in their translational research utility owing to differences from humans in brain structure and function and in drug metabolism. Here, we generated a transgenic marmoset model of the polyQ diseases, showing progressive neurological symptoms including motor impairment. Seven transgenic marmosets were produced by lentiviral introduction of the human ataxin 3 gene with 120 CAG repeats encoding an expanded polyQ stretch. Although all offspring showed no neurological symptoms at birth, three marmosets with higher transgene expression developed neurological symptoms of varying degrees at 3–4 months after birth, followed by gradual decreases in body weight gain, spontaneous activity, and grip strength, indicating time-dependent disease progression. Pathological examinations revealed neurodegeneration and intranuclear polyQ protein inclusions accompanied by gliosis, which recapitulate the neuropathological features of polyQ disease patients. Consistent with neuronal loss in the cerebellum, brain MRI analyses in one living symptomatic marmoset detected enlargement of the fourth ventricle, which suggests cerebellar atrophy. Notably, successful germline transgene transmission was confirmed in the second-generation offspring derived from the symptomatic transgenic marmoset gamete. Because the accumulation of abnormal proteins is a shared pathomechanism among various neurodegenerative diseases, we suggest that this new marmoset model will contribute toward elucidating the pathomechanisms of and developing clinically applicable therapies for neurodegenerative diseases.ArticleeNeuro.4(2):e0250(2017)journal articl

Hsp40 Gene Therapy Exerts Therapeutic Effects on Polyglutamine Disease Mice via a Non-Cell Autonomous Mechanism

<div><p>The polyglutamine (polyQ) diseases such as Huntington’s disease (HD), are neurodegenerative diseases caused by proteins with an expanded polyQ stretch, which misfold and aggregate, and eventually accumulate as inclusion bodies within neurons. Molecules that inhibit polyQ protein misfolding/aggregation, such as Polyglutamine Binding Peptide 1 (QBP1) and molecular chaperones, have been shown to exert therapeutic effects <em>in vivo</em> by crossing of transgenic animals. Towards developing a therapy using these aggregation inhibitors, we here investigated the effect of viral vector-mediated gene therapy using QBP1 and molecular chaperones on polyQ disease model mice. We found that injection of adeno-associated virus type 5 (AAV5) expressing QBP1 or Hsp40 into the striatum both dramatically suppresses inclusion body formation in the HD mouse R6/2. AAV5-Hsp40 injection also ameliorated the motor impairment and extended the lifespan of R6/2 mice. Unexpectedly, we found even in virus non-infected cells that AAV5-Hsp40 appreciably suppresses inclusion body formation, suggesting a non-cell autonomous therapeutic effect. We further show that Hsp40 inhibits secretion of the polyQ protein from cultured cells, implying that it inhibits the recently suggested cell-cell transmission of the polyQ protein. Our results demonstrate for the first time the therapeutic effect of Hsp40 gene therapy on the neurological phenotypes of polyQ disease mice.</p> </div

AAV5-Hsp40 improves body weight loss and extends the lifespan of polyQ disease mice.

<p>R6/2 mice at P7 were injected in both sides of the striatum with AAV5-GFP, AAV5-QBP1, or AAV5-Hsp40, and the following phenotypes were analyzed. (A) Body weight measured weekly. Values represent the mean (*<i>p</i><0.05, R6/2+GFP vs R6/2+Hsp40). (B) Survival (<i>p</i><0.0001, R6/2+GFP vs R6/2+Hsp40, Log-rank test). In both (A) and (B), n≥9 mice.</p

AAV5-Hsp40 also inhibits polyQ inclusion body formation in virus non-infected neurons of polyQ disease mouse brains.

<p>Htt inclusion body formation in 8 week old R6/2 mouse brains injected with AAV5-GFP on one side of the striatum and AAV5-QBP1 or AAV5-Hsp40 on the other side, was analyzed as in Fig. 1, but in the virus non-infected cells on each side of the brain. (A) Representative photographs of striatal (top panels) and cortical (bottom panels) sections of R6/2 mice injected with the indicated viruses. Green; virus infected cells, red; htt, and blue; nuclei visualized by DAPI staining. Representative virus non-infected cells in each panel are indicated by white arrowheads. (B) Inclusion body formation in virus non-infected cells on the AAV5-QBP1 injected side and AAV5-Hsp40 injected side in the striatum (left) and cortex (right). Data are shown as means ± SEM of ≥6 fields of view, in which over 180 cells were counted (**<i>p</i><0.01). Representative results of two mice analyzed are shown.</p

AAV5-Hsp40 improves spontaneous locomotor activity and grip strength abnormalities of polyQ disease mice.

<p>R6/2 mice at P7 were injected in both sides of the striatum with AAV5-GFP, AAV5-QBP1, or AAV5-Hsp40, and the following phenotypes were analyzed. (A) Rotarod performance measured at 4 (left) and 7 weeks (right) of age. (B, C) Open-field activity (B) and rearing (C) measured at 5 (left) and 8 weeks (right) of age. (D) Grip strength measured at 9 (left) and 12 weeks (right) of age. Values represent mean ± SEM, n≥9 mice (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001).</p

Hsp40 inhibits polyQ protein secretion in cultured cells.

<p>(A) Neuro2A cells were co-transfected with plasmids expressing Q81-CFP-V5 and either GFP, QBP1, or Hsp40, and the culture media (CM) and cell lysates (CL) were subjected to Western blot analysis with a V5 antibody to detect the Q81-CFP-V5 protein. (B) Relative amounts of Q81-CFP-V5 secreted into the culture media, calculated from the band intensities in (A), with the amount of Q81-CFP-V5 secreted from cells co-expressing GFP set to 1. (C) Neuro2A cells were transfected with a plasmid expressing Q81-CFP-V5 and siRNA against Hsp40 or a control siRNA, and the culture media (CM) and cell lysates (CL) were subjected to Western blot analysis with a V5 antibody to detect the Q81-CFP-V5 protein, and with an Hsp40 antibody. The loading controls are as in (A). (D) Relative amounts of Q81-CFP-V5 secreted into the cuture media, calculated from the band intensities in (C). In (A) and (C), serum albumin is shown as a loading control for the culture media, and β-actin as a loading control for the cell lysates. In (B) and (D), data are shown as means ± SEM of ≥ four independent experiments (*<i>p</i><0.05, ***<i>p</i><0.001).</p