New research points to two potential approaches for attacking Parkinson’s disease and other synucleinopathies—one going after a well-known culprit, the other targeting an up-and-coming one. In this week’s Journal of Neuroscience, Virginia Lee, University of Pennsylvania School of Medicine, Philadelphia, and colleagues report that shutting down α-synuclein expression reverses synaptic and memory deficits in transgenic mice modeling dementia with Lewy bodies (DLB), even in symptomatic animals. Another study, published online July 5 in the Proceedings of the National Academy of Sciences USA, takes a different tack. Researchers led by Pablo Sardi at Genzyme Corporation, Framingham, Massachusetts, solidify the mechanistic link between synucleinopathies and mutations in the lysosomal enzyme glucocerebrosidase (GBA), and show that boosting brain GBA activity reduces pathology and memory defects in a synucleinopathy mouse model.

For the Journal of Neuroscience study, first author Youngshim Lim and colleagues analyzed mice they had engineered to express inducible mutant (A53T) or wild-type α-synuclein in forebrain neurons (Lim et al., 2010). Transgene expression was kept off from gestation through postnatal day 21 by feeding doxycycline, then turned on by switching the mice to a normal diet. Starting around four months, clumps of α-synuclein cropped up in limbic areas of A53T mice, and intensified with age, much like what appears in brains of DLB or Parkinson’s disease dementia (PDD) patients. The buildup of α-synuclein seemed to correlate with reactive gliosis, a reduction in presynaptic vesicle proteins, and problems with contextual fear memory. Older (20- to 22-month) A53T mice accumulated cytoplasmic α-synuclein and lost hippocampal and cortical neurons. Wild-type α-synuclein mice developed pathology with similar distribution but later onset, and had less severe cell loss than did A53T mice.

Could reducing α-synuclein alleviate pathology and cognitive decline? To find out, the researchers shut off transgene expression in nine-month-old A53T mice. Three months on this diet not only kept α-synuclein from accumulating, but also cleared existing pathology and put a damper on gliosis in the hippocampus. In addition, turning off α-synuclein expression restored synaptic structure and vesicle proteins, and improved memory function.

Robert Edwards of the University of California, San Francisco, found this a “very interesting and important piece of work that will motivate more extensive studies of the synaptic deficits in PD and their potential reversibility.” Subhojit Roy of the University of California, San Diego, was also drawn to the synaptic findings (see full comment below and ARF related news story), and Dimitri Krainc of Massachusetts General Hospital, Charlestown, found the reversibility of α-synuclein pathology compelling. Krainc noted, however, that future therapeutic development requires more research on the mechanism of α-synuclein clearance in these mouse models—in particular, to identify factors that help break up existing α-synuclein aggregates and/or prevent new aggregates from forming (see full comment below).

In the PNAS paper, the Genzyme researchers approach synucleinopathies from another angle. They examined transgenic mice carrying a glucocerebrosidase (GBA) mutation associated with Gaucher's disease. This lysosomal storage disorder has grabbed the attention of PD researchers in large part due to recent work establishing GBA1 as a top genetic risk factor for PD (see ARF related news story; see PDGene). Interest in GBA further intensified with several papers in the past month laying out mechanistically how GBA might influence α-synuclein processing (ARF related news story on Mazzulli et al., 2011 and Cullen et al., 2011). The current study provides further in-vivo support for this mechanistic link, and solidifies the idea that increasing brain GBA may help people with synucleinopathies.

The Eureka moment for first author Pablo Sardi and colleagues was their discovery of α-synuclein aggregates in the brains of GD model mice expressing mutant (D409V) GBA1. Given that PD patients with GBA mutations are more prone to cognitive decline, the team proceeded to look for behavioral deficits in the GD mice. Memory impairment showed up when these animals were put through novel object recognition and contextual fear conditioning tests. The researchers were able to reduce buildup of hippocampal α-synuclein and correct the memory deficit in GD mice by injecting glucocerebrosidase into the hippocampus using adenoviral vectors. So far, the team has shown that this works in mice treated at two months of age and analyzed in behavioral assays two months later. Ongoing studies are testing whether the GBA boost would also benefit older animals with disease well underway, Sardi told ARF.

As for whether the GBA mutation in the GD mice acts through a loss of function or toxic gain of function, the study suggests both play a role. Evidence for the former came from the observation that homozygous GD mice had lower GBA activity and more α-synuclein aggregates than heterozygous littermates, suggesting that loss of GBA function promotes synuclein accumulation. However, the fact that heterozygous GD mice (GBA1D409V/+) do not accumulate GBA lipid substrates, yet do form α-synuclein aggregates, argues that the mutant enzyme may enhance synuclein aggregation through a toxic gain of function. “The take-home message is that both loss-of-function and gain-of-function mechanisms are at play here,” Sardi said. The study also confirms GBA as a promising therapeutic target for treating synucleinopathies (see comments by Valerie Cullen, Joseph Mazzulli, and Dimitri Krainc below).—Esther Landhuis

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Comments on News and Primary Papers

  1. Pablo Sardi and colleagues did an excellent job describing the GBA mouse model; however, the data/experimental design could not decipher the central conundrum, i.e., whether GBA mutations act through a loss of function or a toxic gain of function. The authors speculate that glucocerebrosidase replacement may be a strategy for the treatment of α-synucleinopathies. This is tautology in the paper, but with merit for select patients where GBA mutations can be ascribed as a major contributor to disease.

    Sardi et al. did not address the mechanism in the context of past gene discovery, or the new synthesis emerging. Readers may be aware that we recently identified pathogenic mutations in VPS35, a central retromer component, in late-onset Parkinson's disease. The paper by Vilarino-Guell and colleagues is embargoed in American Journal of Human Genetics until 14 July 2011. My reason for mentioning it briefly is that the retromer is required to recycle mannose-6-phosphate receptors (MPR) that are necessary to traffic lysosomal enzymes, including glucocerebrosidase, from the Golgi complex to an acidified (pre)lysosomal compartment.

    Of note, the endosome Rab7L1 (which we postulate explains the PARK16 GWAS signal) is also required for proper MPR trafficking and for retromer localization and function. Dynactin and tau have also been directly implicated in retromer formation and parkinsonism. α-synuclein has been shown to more generally disrupt cellular Rab homeostasis, in a dose-dependent manner, and may affect multiple trafficking steps between the ER and Golgi. Likewise, LRRK2 GTPase and kinase signalling/scaffolding functions are most consistent with its complex being a master regulator of membrane protein trafficking.

    Many specific details need to be resolved, but GBA, VPS35, Rab7L1, DCTN1, MAPT, SNCA, and LRRK2—indeed most of the major genes identified in late-onset parkinsonism (to date)—now elucidate an overlapping biologic network. The phenomenology of selective vulnerability, variable expressivity, and penetrance may be addressed using a similar framework.

    Ultimately, successful neuroprotective therapeutics for neurodegenerative disorders will result from a combination of genetic insight and model development, and Pablo Sardi's work nicely illustrates the approach.

    View all comments by Matthew M J Farrer
  2. Deciphering details of α-synuclein-induced pathology in the brain has been surprisingly challenging, and the data by Lim et al., 2011, using an inducible system driven by the CamKII-promoter offer significant insights. While there are many notable take-home messages in this exquisitely detailed study, of particular interest to us is the observation that levels of multiple presynaptic vesicular proteins (including transmembrane and peripherally associated proteins) are greatly diminished in boutons overexpressing α-synuclein. The authors posit that this phenomenon is “the likely anatomic substrate of memory dysfunction.”

    As noted by Lim et al., we recently showed that modest increases in α-synuclein levels lead to global decreases in presynaptic vesicular protein levels—a phenomenon we termed "vacant synapses" (Scott et al., 2010). Although this was a straightforward observation of a simple and tractable model system that we generated, it was nevertheless deemed contentious (see ARF related news story). While the exact mechanisms by which excessive α-synuclein leads to decreases in presynaptic protein levels remains unclear, there now seems to be little doubt that reductions in presynaptic protein levels are a bona-fide pathologic consequence of excessive α-synuclein.

    It is interesting to note that several recent studies have implicated excessive α-synuclein at the synapse as the early underlying pathologic substrate in disease. This is in contrast to the longstanding focus on perikaryal Lewy bodies that has dominated the field for decades. Given the unequivocal and robust localization of α-synuclein to presynaptic terminals, the clear induction of synaptic pathology by modest α-synuclein levels, and the relative paucity of somatic Lewy bodies in the vast majority of PD/DLB brains, combined with the presence of presynaptic α-synuclein aggregates in such brains, this seems a logical course of events and perhaps represents a paradigm shift in the field.

    References:

    . A pathologic cascade leading to synaptic dysfunction in alpha-synuclein-induced neurodegeneration. J Neurosci. 2010 Jun 16;30(24):8083-95. PubMed.

    View all comments by Subhojit Roy
  3. This PNAS paper represents the latest in a recent flurry of papers (Xu et al., 2010; Cullen et al., 2011; Mazzulli et al., 2011) examining the biochemical and mechanistic links between GBA mutations and synuclein mismetabolism. In the current paper, Sardi et al. find that mice carrying two copies of the D409V mutation in GBA exhibit progressive mismetabolism of synuclein and generalized ubiquitinopathy.

    We made the same observation in these mice in our recent Annals of Neurology paper (Cullen et al., 2011), where we showed an age-dependent increase in the synuclein content of the membrane fraction (containing within it the lysosomal compartment) and ubiquitin staining. Like Sardi et al., we also observed that mice carrying only one copy of the D409V mutation had subtle changes in synuclein levels, supporting the notion of a gain-of-toxic function as at least one aspect of the mechanistic interplay between GBA and synuclein.

    We also showed an accumulation of synuclein in simple cell models when D409V or the similar variant, D409H, was overexpressed. Importantly, when we overexpressed wild-type GBA in cells, we observed a significant reduction in cellular synuclein levels. This was the case in both PC12 cells, which were transfected with GBA, and in HEK293 cells, which experienced a more robust increase in GBA expression and activity due to viral overexpression.

    Sardi et al. have now extended our observations into animals by showing that viral overexpression of wild-type GBA into rodent brain can reduce the synuclein accumulation and memory deficits caused by GBA mutation. Thus, as discussed in our Annals Neurology paper (Cullen et al., 2011), and as demonstrated by Sardi et al., increasing the brain's GBA content may be a viable therapeutic strategy to explore further. Perhaps eventually, a combination approach may be used, with GBA expression utilized to combat loss of enzyme function, and GBA chaperoning and/or lysosomal support (see, e.g., our results with isofagomine and rapamycin) utilized to combat a gain of toxic function of mutant GBA.

    References:

    . Accumulation and distribution of α-synuclein and ubiquitin in the CNS of Gaucher disease mouse models. Mol Genet Metab. 2011 Apr;102(4):436-47. PubMed.

    . Acid β-glucosidase mutants linked to Gaucher disease, Parkinson disease, and Lewy body dementia alter α-synuclein processing. Ann Neurol. 2011 Jun;69(6):940-53. PubMed.

    View all comments by Valerie Cullen
  4. The paper by Pablo Sardi et al. demonstrates that expression of exogenous glucocerebrosidase (GC) in mice can ameliorate some aspects of pathology observed in Gaucher's disease-like mice. This important finding is consistent with other studies, including our own (Mazzulli et al., 2011) showing that mutant GC contributes to α-synuclein accumulation and pathology. The study by Sardi et al. also suggests that gain of function of GC may play at least a partial role in disease pathogenesis. However, further detailed mechanistic studies will be required to elucidate the relative contribution of this pathway to disease pathogenesis. Importantly, the current study confirms that glucocerebrosidase is an important therapeutic target for the treatment of synucleinopathies.

    The paper by Virginia Lee’s group is an elegant study of reversibility of α-synuclein pathology when expression of α-synuclein is suppressed. This important finding highlights the role of α-synuclein accumulation and clearance in disease pathogenesis. It will be of interest to examine the mechanism of α-synuclein clearance in these mouse models and identify factors that either promote disaggregation of the pre-formed α-synuclein aggregates and/or prevent the formation of new oligomeric or aggregate species of α-synuclein. Identification of such factors that ultimately mediate removal of excessive α-synuclein from affected cells would facilitate the development of new therapies for PD and related synucleinopathies.

    The first example of reversibility in accumulation and toxicity of aggregation-prone proteins has been shown in mouse models of Huntington’s disease (Yamamoto et al., 2000), and later, Harry Orr’s group demonstrated partial reversibility in mouse models of SCA1 (Zu et al., 2004). In addition, a similar phenomenon was observed in models of prion disease (Mallucci et al., 2003). Together with the nice study by Dr. Lee, these studies suggest that various forms of aggregation-prone proteins, such as monomers, oligomers, and aggregates, exist in some sort of equilibrium if the production of the monomeric form is maintained. However, when expression of monomeric forms is suppressed, these other moieties are amenable to degradation by cellular degradation machinery. Further mechanistic dissection of these pathways will be critical for future therapeutic development.

    References:

    . Gaucher disease glucocerebrosidase and α-synuclein form a bidirectional pathogenic loop in synucleinopathies. Cell. 2011 Jul 8;146(1):37-52. PubMed.

    . Reversal of neuropathology and motor dysfunction in a conditional model of Huntington's disease. Cell. 2000 Mar 31;101(1):57-66. PubMed.

    . Recovery from polyglutamine-induced neurodegeneration in conditional SCA1 transgenic mice. J Neurosci. 2004 Oct 6;24(40):8853-61. PubMed.

    . Depleting neuronal PrP in prion infection prevents disease and reverses spongiosis. Science. 2003 Oct 31;302(5646):871-4. PubMed.

    View all comments by Dimitri Krainc
  5. The paper by Sardi et al., 2011, provides further important in-vivo evidence of a mechanistic link between Gaucher’s disease and α-synuclein processing. The demonstration that accumulation of α-synuclein and behavioral deficits in Gaucher's disease mice can be ameliorated by increasing glucocerebrosidase levels suggests that this lysosomal enzyme may be an important therapeutic target for the treatment of synucleinopathies. It will be of future interest to determine whether enhancing glucocerebrosidase function, either through adenoviral-mediated glucocerebrosidase expression or administration of pharmacological chaperones, has the ability to reverse or clear the accumulation of α-synuclein in aged (12-month-old) Gaucher's mice that are symptomatic.

    The recent exciting paper by Lim et al. (Lim et al., 2011), which demonstrates that behavioral deficits and pathology induced by α-synuclein overexpression can be reversed, suggests that methods that enhance the clearance of α-synuclein may provide therapeutic benefit even after symptoms are apparent. Augmentation of glucocerebrosidase function appears to be one such option that may accelerate the clearance of α-synuclein and prevent further disease progression in Parkinson's disease and other synucleinopathies.

    References:

    . α-Syn suppression reverses synaptic and memory defects in a mouse model of dementia with Lewy bodies. J Neurosci. 2011 Jul 6;31(27):10076-87. PubMed.

    View all comments by Joseph Mazzulli

References

News Citations

  1. Excess α-Synuclein Sends Synapses Sputtering
  2. More Than Gaucher’s—GBA Throws Its Weight Around Lewy Body Disease
  3. Feedback Loop—Molecular Mechanism for PD, Gaucher’s Connection

Paper Citations

  1. . Forebrain overexpression of alpha-synuclein leads to early postnatal hippocampal neuron loss and synaptic disruption. Exp Neurol. 2010 Jan;221(1):86-97. PubMed.
  2. . Gaucher disease glucocerebrosidase and α-synuclein form a bidirectional pathogenic loop in synucleinopathies. Cell. 2011 Jul 8;146(1):37-52. PubMed.
  3. . Acid β-glucosidase mutants linked to Gaucher disease, Parkinson disease, and Lewy body dementia alter α-synuclein processing. Ann Neurol. 2011 Jun;69(6):940-53. PubMed.

External Citations

  1. PDGene

Further Reading

Papers

  1. . Gaucher disease glucocerebrosidase and α-synuclein form a bidirectional pathogenic loop in synucleinopathies. Cell. 2011 Jul 8;146(1):37-52. PubMed.
  2. . Acid β-glucosidase mutants linked to Gaucher disease, Parkinson disease, and Lewy body dementia alter α-synuclein processing. Ann Neurol. 2011 Jun;69(6):940-53. PubMed.
  3. . Accumulation and distribution of α-synuclein and ubiquitin in the CNS of Gaucher disease mouse models. Mol Genet Metab. 2011 Apr;102(4):436-47. PubMed.
  4. . Forebrain overexpression of alpha-synuclein leads to early postnatal hippocampal neuron loss and synaptic disruption. Exp Neurol. 2010 Jan;221(1):86-97. PubMed.

Primary Papers

  1. . α-Syn suppression reverses synaptic and memory defects in a mouse model of dementia with Lewy bodies. J Neurosci. 2011 Jul 6;31(27):10076-87. PubMed.
  2. . CNS expression of glucocerebrosidase corrects alpha-synuclein pathology and memory in a mouse model of Gaucher-related synucleinopathy. Proc Natl Acad Sci U S A. 2011 Jul 19;108(29):12101-6. PubMed.