A Novel Protein Complex Linking the Glutamate Receptor and Autophagy: Implications for Neurodegeneration in

Lurcher Mice

Yue, Horton, Bravin, DeJager, Selimi and Heintz.

Neuron, Vol. 35, 921-933, 2002.

Lurcher mutant mice

• First described in 1960

• Heterozygous lurcher mice are ataxic (irregular, uncoordinated movements) due to degeneration of the cerebellar cortex during the first 4 postnatal weeks

• 1997: Defect is due to constitutive activation of the 2 Glutamate Receptor (GluR2Lc); causes an inward current (of Na+ and possibly Ca++) and subsequent death of Purkinje cells that is cell autonomous

• Secondary death of cerebellar granule cells and inferior olivary neurons follows Purkinje cell death

• Used as a model for studying neurodegeneration in vivo

The human brain

Frontal Lobe

Temporal Lobe

Parietal Lobe

Occipital Lobe

Pons

Medulla

Layers of the Cerebellum

Mechanisms of Purkinje cell death

• 1995, 2000: Apoptotic pathways are activated in both Purkinje cells and granule cells in lurcher animals

• 2000: Bax inactivation (inhibits apoptosis) in lurcher mutants rescues cerebellar granule cells but not purkinje cells

– Apoptotic pathways play a minor role

– Death of cerebellar Purkinje cells must also involve other pathways (Bax independent)

2002 (this paper) : identify signaling pathways that may be associated with the GluR2 receptor and whose constitutive activation might contribute to Purkinje cell death in Lurcher mice

nPIST interacts with the GluR2 C terminus

Yeast 2 H identified nPIST; identical to PIST (PDZ domainprotein interacting specifically with TC10, a Rho family member) except for the addition of 8 aas

A. nPIST: PDZ domain + 2 coiled-coil domains

Q: Is nPIST differentially expressed?B. RT-PCR of PIST and nPIST: nPIST is enriched in whole brain and cerebellum (and heart!)

Q: Is nPIST regulated similary to GluR2?C. Western blot anlaysis of expression during postnatal development: expression of both proteins increases as the cerebellum maturesD. Fractionation of cerebellar extracts: nPIST cofractionatedwith GluR2 in cerebellar extracts and was similarly enriched In the synaptosomal and PSD (postsynaptic density of Purkinjecell:parallel fiber synapses).

Interaction between GluR2 and nPIST is specific

Q: Can nPIST and GluR2 interact in mammalian cells?A. Flag-tagged FL nPIST and C term of GluR2 Coimmunoprecipitate in HEK293 cells (human embryonic kidney)

Q: Is the interaction specific?B. Yeast 2H: Interacts with both GluRd2 and its close relativeGluRd1; requires the last 3 aas of these receptors; No interactiondetected with GluR2, another receptor on Purkinje cells

Q: What domains are required for nPIST to bind to GluR2 In mammalian cells?C. ConstructsD. Did Co-Ips with FL-Flag-GluR2 and various GFP fusion constructs- Showed requirement for PDZ domain

Colocalization of nPIST with GluR2 requires the nPIST PDZ domain

Confirmation of co-IP experiments:Direct visualization of colocalization ofGFP-nPIST derivatives and GluR2 in transfected HEK 293 cells.

nPIST coiled-coiled domain interacts with Beclin1

Q: Is nPIST acting as an adaptor protein?nPIST coiled-coil domains highly conserved with C.elegans nPIST; conducted Yeast 2H using the nPIST coiled-coil domains and identified Beclin-1

A. Schematic of Beclin-1: Bcl-2 BD and coiled –coil domain (ortholog of yeast Apg6)

Q: Is the interaction specific?B. GST pull-down assays (in vitro translation reactions):nPIST coiled-coil + Beclin 1 interact

C. Co-IPs in HEK 293 T cells:FlagnPIST + Beclin1-myc form a complex in mammalian cells.

nPIST coiled-coiled domain interacts with Beclin1

More interaction assays:D. nPIST and Beclin 1 colocalize in HEK 293T cells;Colocalization occurs in perinuclear area (inset), consistent with reports that Beclin 1 is present in the trans-Golgi network.

Q: Do nPIST and Beclin1 colocalizein the cerebellum?E. Both nPIST and Beclin1 present in thecell bodies & dendrites of cerebellarPurkinje cells

Q: Do GluRd2, nPIST and Beclin1 forma complex in vivo?E. Did co-IPs from cerebellar extracts using a GluRd2 antibody; both nPISTAnd Beclin1 Co-IP’d.

Beclin-1 redistributes in cells after induction of autophagy

Q: Is the role of Beclin 1 in autophagy modulated by nPISt?

First wanted to develop a rapid assay for autophagy; prepared a Beclin1-GFP fusion andUsed it to visualize autophagy in nutrient-deprived cells.A. Control cells: Beclin1 is localized to the perinuclear area (trans-Golgi).B. Nutrient deprived cells (no culture medium):Beclin1 redistributes to large vesicular structures.

Q: Does this redistribution reflect activation of autophagy?C and D: vesicular structures (autophagosomesAnd autophagolysosomes) stained positive forBeclin1E: Forming autophagosomes stained positive for Beclin1.

The coiled-coil domains of nPIST and Beclin1 act synergistically

Full-length nPIST and Beclin1 cotransfection:Only about 12% of cells exhibit vesicular staining (B).Q: Is the interaction of nPISt and Beclin1 regulated through the PDZ domain?

B. Deleted PDZ domain and saw vesicular Beclin1 staining in 45% of transfected cells. Under starvation conditions thisincreased to 55%.

Conclude: interaction of nPIST with Beclin1 through its coiled-coil domain can modulate Beclin1 activity.

A. Arrows show vesicular colocaliztion.

GluR2Lc-mediated cell death in HEK 293 cells involves autophagy

Q: Does the constitutive activation of GluR2Lc serveas a stimulus for autophagy and does this contribute toGluR2Lc-mediated death in vitro?

A. Cells expressing activated GluR2Lc contain vesicular Beclin1, but those expressing the wild-type receptor do not.

B. Quantitation of results in A. Constitutive activation of the receptor resultsin a strong induction of autophagy.

C. Does induction of autophagy correlate withcell death?Observed increase in dying cells in Glu2Lc

transfected cells compared to WT transfectedcells. Also, death of Glu2Lc cells was decreased in the presence of 3-methyladenine(3-MA), an inhibitor of autophagy (*also apoptosis).

Purkinje cells from lurcher mice contain autophagic structures

Q: Is autophagy activated in dying lurcher Purkinje cells?

Previous EM studies indicated anincreased number of lysosomes, swollen mitochondria and other Unusual features but autophagicstructures not specifically reported.

So did additional EM: Autophagosomes (A,B,C) and Autophagolysosomes (D) were frequently observed.

Autophagosomes were not observed in wild-type samples.

Summary/Discussion

• identified protein interaction between C terminus of GluRd2 and the PDZ domain of nPIST

• Identified protein interaction between the coiled-coil domains of nPIST and Beclin1

• In the absence of its PDZ domain, nPIST acts synergistically with Beclin1 to induce autophagy; both proteins are present in autophagic vacuoles

• GluR2Lc, but not WT, is able to induce autophagy (suggest that this results from the release of Beclin1 or Beclin1+nPIST from the receptor complex)

• data demonstrate the activation of autophagy in dying lurcher Purkinje cellsand suggest that this process may contribute to lurcher-mediated cell death in vivo

• Need to do loss-of-function studies: e.g. if you inhibit autophagy (e.g. knock-outBeclin1), do you inhibit Purkinje cell death??

• Do nPIST and Beclin1 complex with other cell surface receptors?• Is there an nPIST in Drosophila?• Tools and in vitro cell systems for studying autophagy/cell death (Qadir)