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| Hereditary Disease
Disposing of Cellular Garbage: Implications
for Neurodegenerative Diseases
July 19 and 20, 1998
Playa Del Rey, California
Prepared by Rati Verma
HEREDITARY DISEASE FOUNDATION
Disposing of Cellular Garbage : Implications for Neurodegenerative Diseases
Progressive neurodegenerative disorders such as Huntington's disease (HD) and
Spinocerebellar ataxia type I (SCA1) are caused by CAG/polyglutamine (polygln) repeat
expansions in Huntingtin and Ataxin-1 respectively. Besides these two, four other
neurodegenerative diseases have been characterized where these expansions have
also been observed. The mechanism by which the presence of these repeats leads to
neurodegeneration is currently unknown. However, the pathogenesis of both these
diseases has several aspects in common. The brains of affected patients as well as
transgenic mice display neuronal intranuclear inclusions (NII) which accumulate
aggregated Huntingtin and Ataxin-1. The inclusion bodies also stain immunopositively
for polyubiquitin as well as subunits of the proteasome (Cummings et al., Davies et al.).
The 26S proteasome is a highly specialized degradation machine designed to degrade
proteins in an ATP-dependent fashion. It is comprised of 19S regulatory caps that seal
off the inner 20S protease core at both ends from the cellular milieu. Proteins targeted
for degradation by the 26S proteasome are multiubiquitinated, receptors for which exist
in the 19S caps. The sequence of steps leading to recognition and degradation of the
multiubiquitinated protein are currently not well characterized, although it is well
established that the multiubiquitinated protein is degraded in a processive fashion. Co-
immunostaining of polyubiquitin and Huntingtin/Ataxin-1 has led to the speculation that
these proteins could be polyubiquitinated but not degraded. The 26S proteasome could
thus be competitively blocked, thereby disrupting normal nuclear function, leading
eventually to neurodegeneration. It was also found for ataxin-1 that over-expression of
the cellular Chaperone HDJ-2/HSDJ decreased the frequency of Ataxin-1 aggregation,
leading to the speculation that the presence of the polygln tracts leads to misfolding and
Since the new studies implicated both proteasomes as well as chaperone
proteins in the pathogenesis of the neurodegenerative diseases, expert scientists from
both fields got together with the scientists actually studying the diseases for a lively
discussion lasting two days. The session concluded with each participant suggesting an
experiment in her/his field of specialty designed to further advance our understanding of
the neurodegenerative diseases.
The session opened with a mother introducing her 22 year old daughter suffering
from HD. The father, who had been diagnosed with the disease at 33, had passed
away at the age of 42. The patient has three siblings :- two sisters and one brother.
The oldest sister is still asymptomatic. The second has tested positive while the brother
has elected not to get tested. The patient was being treated with a drug called Riluzole.
Riluzole appears to inhibit glutamate release and blocks excitotoxicity. It prolongs the
life of SOD transgenic mice and may also extend the lives of some ALS patients.
Nancy elaborated on the symptoms of this young woman who manifests symptoms
more characteristic of the juvenile form of the illness. She shows extreme stiffness and
slowness, as opposed to involuntary movements in all parts of the body, which marks
the adult form so dramatically. The young woman also suffered impaired coordination
and speech, loss of rhythmicity and rapidity of movements and speech. Predictive
testings is now available for HD. Only 5-10% of the people who could take the
predictive test have, in fact, taken it. According to a recent worldwide survey of testing
centers, testing has a 1% morbidity and mortality rate.
Goldberg asked the question as to how well could early and late onset HD be
compared? He also commented that the patient's fine motor control seemed more
affected than coarse control.
Schwartz replied that fine muscles have more innervation.
Question Does HD arise as a consequence of inappropriate turning on of the cell death
machinery or does abnormal cellular function arising from the aggregated Huntingtin
protein eventually lead to cell death?
Davies : A transgenic mouse line has been generated that displays many of the
symptoms displayed by patients suffering fromHD. In these mice, exon 1 of human
Huntingtin carrying CAG repeat expansions of 115-156 units is expressed under the
control of the human promoter. The mice (Bates mice) have symptoms and smaller
brains well before cell death. Also no TUNEL positive cells were detected. No gliosis,
inflammation nor astrocytosis was detected. The status of p53 was not investigated.
Cummings : Agreed that the SCA1 disease model shows no signs of apoptosis.
However, astrocytosis has been observed.
Davies: The mice are better models of juvenile rather than adult onset HD. As in
juvenile HD, these mice show effects on Purkinje cells and suffer from seizures..
DeFiglia was quoted as saying that the Purkinje cell effects are a consequence of the
The different mouse cell lines such as R6/1, R6/2, R6/5 have the same mRNA levels
but different stages of onset. This is probably a reflection of the protein level because
the R6/5 homozygote shows symptoms much earlier than the heterozygote. The
symptoms (observed only for Q 140 mice, but not Q18 mice) are: Presence of inclusion
bodies. They first appear in the cortex, then the striatum, then progressively in other
areas of the nervous system. The inclusion bodies are also observed in dystrophic
neurites (myelinated and non-myelinated dendrites) and glia. The latter are fewer in
number but display more uniform staining. The inclusions are more nuclear in juvenile
onset and more neurite in late onset HD. They are not detected in non-neuronal
tissues, except in muscle, that too only in mice but not in humans.
Antigen Immunostaining. Several antibodies (Ab) to Huntingtin (htt) were described.
Li's Ab EM48 is a polyclonal antibody made against the amino-terminus of Htt lacking
the poly-Q region. Ab EM48 is a polyclonal antibody made against the amino-terminus
of Htt lacking the poly-Q region. This antibody preferentially stains the abnormal
huntingtin as clumps/aggregates in the cytosol and the nucleus. These clumps are
observed early on, but interestingly, not in cells that are spared later on.
The Cardiff group has about 25 different antibodies to regions downstream of 500 aa
which do not detect the protein suggesting that truncation of Htt is an early event. The
IC2 (Ab to poly Q) did not immunoreact although Fischbeck has developed conditions
where this antibody does stain
Proteasome immunostaining. Proteasomes are recruited late to the inclusion bodies,
but once detected, their staining is quite uniform. The 20S core subunits stain nuclei
strongly in both normal and HD. The 19S regulatory cap subunits TBP7 and P31
staining is not as uniform as the 20S core. The 11S components such as PA28 alpha
stains all inclusions strongly. The beta Ab never stains anything in the nucleus while Ab
to the gamma subunit never stains inclusions. Ki, which is a strong nuclear antigen,
never stains inclusions. Ubiquitin was also detected. The antibody used detects only
ubiquitin conjugates, not free monomeric ubiquitin. The ubiquitin-like homolog-SUMO1
was not detected. Neither was PML protein. Neither were E2-25K (HIP2) nor PDP9.5
The deubiquitinating enzyme UCHL3 was also detected. The status of E1, the enzyme
that catalyses the first step of ubiquitin activation, was unknown.
Vacuolization : Cells are spongiform at the terminal stages indicating degeneration.
Questions raised at the end of the description of the mouse model of the disease
voiced concern over technical issues such as 1. If 20S staining was uniform, why wasn't
19S detected uniformly? 2. How good were the antibodies? 3. Did the aggregated
structures somehow prevent staining of the 19S? 4. Was there non-specific trapping of
proteins in the inclusion bodies? 5. Since the 11S PA28 complex is very stable, how
come PA28-alpha was detected but not the PA28 beta or gamma subunits? 6. It has
not been formally shown that Htt is polyubiquitinated. Might some other component be
polyubiquitinated? Tau is only monoubiquitinated. 7. Inclusion bodies-material is hard
to come by in purified form because of low amounts and insolubility.
Cummings gave an update on the studies done on spinocerebellar ataxia type 1
(SCA1). This is a neurodegenerative disorder caused by expansion of a polyglutamine
tract in ataxin-1. Unlike HTT, which is a large protein of 350 kDa, this protein is only
100 kDa. The SCA1 transgenic mice must express high levels of the expanded polygln
(Q82) protein to display early pathological symptoms. The protein is expressed under
the Purkinje cell promoter. Wild-type ataxin-1 is cytoplasmic in peripheral cells and
nuclear in neuronal cells. The Q82 animals form nuclear inclusions that are small and
punctate at first. As the disease progresses, they seem to coalesce. The inclusions
are immunopositive for polyubiquitin, 20S and 19S (P31). No immunoreactivity was
detected with Hsp70 antiserum. Instead, low levels of hsc70 were detected, in some
inclusions only. Cummings also made the point that neuronal dysfunction was observed
before the appearance of inclusion bodies. So the question remains if they are the
cause or the effect of the disease. Mice transgenic for SCA-1 with the nuclear signal
mutated display no behavioral abnormalities. Consistent with the lack of symptoms, the
protein is cytoplasmic and no inclusion bodies are observed.
The Q30 animal also makes small punctate nuclear inclusions.
HeLa cells were transfected with ataxin-1 (Q2 and Q82), under the control of the CMV
promoter. Inclusions were observed in 5 % of the cells expressing ataxin-1 with Q2 and
Ú75% of cells expressing ataxin-1(Q82). The inclusions were immunopositive for
polyubiquitin, 20S, HSDJ2 (DnaJ), HSP70, Hsc70. Co-transfection of mutant ataxin-1
and Hsp70 (both under the CMV promoter) showed that HSP70 colocalized with ataxin-
1 in both punctate and large inclusions, but no consequent reduction in frequency of
inclusions was observed. The inducible isoform was used, although it was not formally
proved that it was being induced as expected. In contrast, over-expression of HSDJ2
caused a shift to smaller punctate structures; the frequency was decreased two fold.
PML bodies redistributed with ataxin-1. Colocalization of the deubiquinating enzyme
was observed (HAUSP).
Work in progress-An inducible promoter would be used to follow ataxin-1 aggregation
and to study the roles of chaperones and the proteasome in SCA1. Transgenic mice
were being generated that would express Wildtype and Mutant HSDJ2 under the
Purkinje cell promoter.
Follow up questions/comments.
Goldberg, Baumeister, Schwartz : About 78% of the cell's proteasomes are in the cell
nucleus. Goldberg had the question whether it was the constitutive or inducible form of
the HSP that was colocalized to the inclusion body?
Mayer : Proteasomes are localized on nuclear envelopes. Observed protein
precipitation in all classes of neurodegenerative diseases.. All the precipitated proteins
were ubiquitinated. The question is, why does it take so long for HD to develop if
protein aggregation is an early event?
Goldberg, Ciechanover : Many hemoglobinopathies result in abnormal globins that
aggregate and form aggregates. The proteasome can clear these aggregates. The
Alpha-1 antitrypsin "z" variant makes inclusions in the ER
Horwich, Goldberg : A kinetic description, i.e. a pulse chase analysis of Htt fate needs
to be determined. Is there a fraction of Htt that is not degraded? Are the initial
aggregates cytosolic? The Bonini group (Cell paper, Warrick et al.) has shown that
non-neuronal aggregates are not toxic. Is that because of clearing and degradation?
Are inclusions formed because production outpaces degradation? In E.coli, do
chaperones keep Htt soluble or are they involved in translocating it to the proteases?
In E.coli, inclusions generally disappear during stationary phase.
Hartl noted that Lindquist has shown that chaperones can solubilize protein aggregates
Horwich : Pathology may precede inclusions.
Baumeister : When does ubiquitination occur relative to aggregation? Further
biochemical analysis is needed.
Schwartz: Noted that quantitative histology of disease is lacking-i.e the size and
number of neurons affected should be quantitated. Described the characteristics of
short term memory in Aplysia. Increased cytoplasmic phosphorylation blocks a
potassium channel. This delays repolarization which increases neurotransmitter
output for 5-10 min. Long term memory : Protein kinase A is translocated into the
nucleus where it phosphorylates CREB which increases gene expression. Long
term facilitation : CREB upregulates ubiquitin C-terminal hydrolase which
activates proteasome. The pKA regulatory subunit is degraded, leaving the catalytic
subunit active. Phosphorylation persists for 24 hr. Blocked by proteasome
inhibitors (MG132, lactacystin). Facilitation is also observed by injection of catalytic
subunit (but it not blocked by proteasome inhibitors)
Wilkinson : DOA4, UCH : These are enzymes that deubiquitinate polyubiquitinated
proteins. One of these, Isopeptidase T is the last step in deubiquitinylation. Mentioned
a developmental checkpoint in Dictyostelium where if you block degradation, there is a
failure to form slug (cAMP dependent).
Schwartz : made the made the point that flow could be regulated by degradation
whereas specificity could be regulated by ubiquitin conjugation
Pickart : Observed that a pile up of ubiquitinated proteins can competitively inhibit
Ciechanover : Observed that the inhibitory effect of repeats within proteins on
degradation mediated by the proteasome may be general. Described the gly repeat
in p105 which blocks degradation midway generating the p50 derivative. The Gly-
ala repeat in EBNA-1 probably makes it inaccessible to the proteasome even
though the protein is still conjugated to ubiquitin. It is possible that repeats protect
the protein molecule from degradation wherever it is in the sequence. Maybe there
is less Polygln degradation due to proteasome depletion? Could label with 35S-met
and follow the kinetics of degradation.
Goldberg : Asked the question as to what's rate limiting in the pathway? Global
acceleration of degradation rate can occur without increase in number of
proteasomes. The question is, can Htt saturate the proteasomes in vivo? Also
mentioned data showing that inhibition of proteasomes with MG132 or lactacystin
turns on the heat shock response. This raises the possibility that monitoring the
induction of the heat shock response would be a way of assessing if proteasome
function is blocked due to accumulation of polygln expansion proteins. Also
mentioned that MG132 & lactacystin block apoptosis in neuronal cells (EMBO
paper). It is also possible that these proteasome inhibitors may induce apoptosis in
dividing cells since these compounds are more cytotoxic to cancerous than normal
cells. ProScript (a Biotech company in Boston) has filed an IND for these
compounds for cancer. Also see Tanaka & Nomura in latest BBRC.
Cummings : Mentioned that treatment of HeLa cells over-expressing Ataxin-1 with
MG132 & lactacystin gives more inclusions. Could be a small differential effect? In
the mouse model, hsp70 & HDSJ don't appear upregulated. However, since
Purkinje cells are only 0 1% of cerebellum, the upregulation could have been
Schwartz : Could constipated proteasomes be depleting cells of ATP?
Goldberg : The stress (HS) response is triggered by abnormal proteins. Hsp90
monitors this response while Hsp70 upregulated as a consequence. The question
is, can HD cells mount this response? Also mentioned that the HS response falls off
with aging (Cold Spring Harbor book on stress response). No data is available for
proteasomes in aging
Hochstrasser : Hsp70 interferes with Drosophila development but didn't know what
the HS sensor is.
Mayer : Mentioned that Hsp70 is upregulated in scrapie mice. Treatment of mice with
griseofulvin resulted in an increase in polyubiquitination followed by a subsequent
decline and accumulation of aggregates. In hepatocytes in culture (not in liver),
keratin aggregates disappear in a membrane process. Mark Shearman has a MS
system combined with Ab capture of substrate (SMALDI) that may be a useful
technique for studying substrate changes (address: Merck Sharpe & Dohme,
Neurosciences, Terlings Park, Harlow, UK). Also see Ihara paper (ubiquitin and
Haas : The role of ubiquitin conjugation in pathogenesis of HD should be assessed.
The fact that different areas of the brain are affected could be explained by
differential sequestration of conjugating enzymes. The levels of E1 & E2s in
different sections of the brain could be looked at by immunohistochemistry.
Davies : Michel Goedert (Cambridge UK): a-synuclein, Lewy bodies, etc.: clarified
sarkosyl extract gives dispersed filaments. This is not true for HD.
Wilkinson, Haas : Described a reaction that was enzyme independent and would
result in isopeptide bond formation. This reaction could result in intrachain
crosslinks. The reaction would be sensitive to hydroxylamine in base. (gln-gly
dipeptide; gln side chain bond tautomerizes to 6-membered imine ring; breaks
chain; yields D-glu).
Cummings : Don't find transglutaminase in SCA-1 inclusions
The day started with a review on Chaperones by Hartl.
The major function of chaperones is to prevent misfolding and aggregation of newly
synthesized proteins as well as proteins denatured by stress. The chaperones
recognize exposed hydrophobic residues that are normally buried in folded proteins.
The Hsp70 class of chaperone proteins bind the exposed hydrophobic residues of
unfolded proteins with concomitant hydrolysis of ATP and transfer of conformational
changes to binding domain. The result is a productive folding reaction. On
ribosomes, they stabilize the conformation of the newly synthesized protein until the
entire folding domain emerges.
Chaperonins : They are large cylindrical protein complexes that encapsulate &
sequester misfolded polypeptides. They do not recognize folded polypeptides
because the hydrophobic residues are buried. They do not function catalytically.
Rather, they shift the equilibrium to a productive folding pathway by blocking off
other pathways. Chaperones usually don't act alone but cooperate with cofactors
that regulate ATP exchange and stimulate ATP hydrolysis. They typically don't
resolve preformed aggregates. The only known exception is Hsp104 in yeast. It
solubilizes what may be loose aggregates.
Questions : Can chaperonins prevent the aggregation of Htt? Could they be
interacting with htt on the ribosome?
Horwich : Remarked that it is not clear if there is a human homolog of Hsp104. Also
noted that the polygln domain is at the N-term of htt; synthesizing the whole protein
would take several minutes.
Baumeister : Noted that there is a large body of literature describing the behavior of
polygln polymers at water-air interfaces. These studies involved infrared
spectroscopy of monolayers, and were published primarily in journals such as
Davies, Goldberg, Horwich : Follow up Questions : Would truncated and full length
Htt proteins have the same interaction with chaperones? And with ubiquitin? Does
the presence of expanded polygln repeats change the ubiquitination pattern of the
truncated exon 1 of Htt? Is polygln a substrate for a protease? The need for model
studies studying the effects of polygln expansions on proteasome function were
Haas : Could the C-terminal cleavage of htt occur after it's aggregated?
Hartl : Hsp90 may be the regulator of the induced stress response to aggregated
protein. Described the antibiotic geldanamycin (in preclinical trials for inhibition of
tyrosine kinase activity) which binds to Hsp90 and inhibits its function. Hsp90 is
needed for the correct folding of tyrosine kinase receptors.
Goldberg : StressGen (a company) is trying to induce the heat shock response
therapeutically. Proteasome inhibitors also induce the HSP response. Takeshi
Yura et al (Kyoto) are generating agents that induce the heat shock response.
Ciechanover : Spergolin & deoxyspergolin bind to Hsp70.
Hartl : Couldn't reproduce that, no specific effect.
Horwich : Bristol-Myers Squibb was working on this but they stopped.
Mayer : Mentioned a recent paper by Rick Morimoto in PNAS. This paper studies the
induction of HS response in different brain areas.
Goldberg : Described the existence of four heat shock transcription factors that may be
involved in the induction of the HS response. The differences between the four were
not clearly defined. Some were induced by growth factors because they have the
early response elements in their promoters in addition to the heat shock response
Davies : Earliest change in mice: small dot <1m, stain for ubiquitin about 1 week later.
Question : What would happen if there are two different triplet repeats in the same
animal/ Or even the same cell?
Cummings : Tagged polygln will colocalize with Ataxin-1 as aggregates in nuclear
inclusions in cotransfected HeLa cells.
Goldberg : Suggested the use of polygln to target a protease to mutant htt. Also
emphasized that studies of structure of the aggregates should be a priority.
Pickart : Described her studies with Htt containing polygln repeats varying from 30 to
120. These proteins were in vitro transcribed and translated in reticulocyte lysates.
Translation was normal, with no aggregation. No degradation of the ubiquitinated
proteins by the proteasome was observed.
Gorbea : Described his observation that addition of reticulocyte lysates to insoluble
aggregates made in E. coli, causes them to solublize to monomers.
Schwartz : Could second messengers be damaged by polygln? Suggested that
response to serotonin be checked.
Mayer : Test this in mice?
Davies : LTP & LTD in Bates mice being looked at by Steve Dunnett
Mayer : Highlighted the need to be cautious concerning the localization of inclusions.
In Japan, where patients are kept alive longer, inclusions are found in the motor
cortex. Also described Prusiner's observations that membrane bound prion blocks
secretory pathway but no inclusions are formed.
Cummings : SCA-1 knockout is not embryonic lethal like htt knockout.
Pickart, Wilkinson : Suggested making N-term htt with a ubiquitinatable tag to look at
proteasome activity in vitro
Ciechanover : Observed that fundamental information was needed to evaluate the
pathology of the disease. In the HeLa cell model, to what extent is the protein
ubiquitinated? Does the level of ubiquitinated protein exceed that of the
Hartl : Can delete chaperones from the extract with Ab, then add them back.
Horwich : Need in vitro endpoint that matches in vivo endpoint.
Baumeister : Don't need much material to study architecture of inclusions.
Gorbea: Balance of production & degradation : are wild type & mutant htt made at
Haas : Stressed the age of onset issue. Is there a partitioning of a minority population
of htt to a special form? Question is, would a small accumulation of Htt, over
decades, lead to nuclear aggregation and cytotoxicity?
Pickart : Citing the Warrick et al Cell 1998 paper using Drosophila as the model
system, said that inclusions by themselves were not cytotoxic.
Tobin : Suggested looking at chaperones in the fly system.
Horvich : Hard to get small molecules in Drosophila, since they have no circulation and
their larvae are relatively impermeable.
Wilkinson : Described the construction of stable cell lines (COS) made with GFP
reporter constructs designed for studying the activity of the ubiquitin-dependent
degradation pathway. The reporter constructs were targeted to the cytoplasm or
nucleus (plus/minus nuclear localization signals). These cell lines could now be
transiently transfected with polygln containing constructs.
Schwartz : Dopaminergic neurons accumulate synaptic vesicles with age. Membranes
form inclusions. Except for maybe sickle cell, is there any cell where aggregates are
known to be toxic?
Cummings : HeLa transfection : unlike HD & AR, SCA-1 expression is not toxic.
Hartl, Haas, Verma, Ciechanover : Observed that besides studying normal and
abnormal Htt in cell lines, the proteins could also be studied in vitro. Bacculovirally
produced proteins could be ubiquitinated in vitro and presented to purified
proteasomes. The insect cells could also be used to follow the rate of
synthesis/degradation of normal and mutant Htt by metabolic labeling.
Goldberg : Described Leslie Orgel's "error catastrophe" theory : All proteins are
synthesized with a finite error rate. Since the error rate is finite then each error
increases the chance of another error. He showed that the description of the
accumulated error gives an expanding Taylor series which should predict death. As
such, it is used as a model for aging. However, this theory suffers from the
assumption that all proteins are long-lasting. If one could build in assumptions
about degradation, turnover, etc, then could the theory be used to model age of HD
Hartl : Chaperones are found in nucleus as well as cytoplasm
Haas : Emphasized the idea that different areas of the brain are differentially
permissive to the presence of the polygln containing expansion protein. He would
therefore look for differences in the ubiquitination pathway responsible for degrading
the ubiquitinated Htt. For instance, he could look at immunohistochemistry of E1
and E2s in different brain regions.
Horwich : Does clipping of Htt occur before or after proteasome interaction?
Mayer : 1. Determine which protein in the inclusions is ubiquitinated.
2. Look for "peripherin", a neurofilament protein expressed in response to injury.
3. Is the polygln expansion protein phosphorylated? Or post-translationally modified in
4. Are inclusions inducible? Do they go away?
5. Look at distribution of heat shock proteins in the brain.
6. General Comments : A neuron-specific DnaJ protein has been published. Also, the
example of the Sendai protein may be relevant: When Sendai is expressed in cells,
it ends up perinuclear by virtue of the lysosome system. It is possible that maybe
early on the expanded polygln proteins are cleared by lysosomes. When they fail,
ubiquitination takes over
Davies : With new Ab (EM48, see above) sees htt in multivesicular bodies. Possibly
increased cytoplasmic staining in lysosomes.
Ciechanover : Suggested performing pulse chase studies with immunoprecipitation.
Lysosomes could be selectively inhibited with NH4Cl or leupeptin, while
proteasomes could be blocked with lactacystin. Thus it could be determined which
pathway leads to formation of inclusions (but note ; monoubiquitinated proteins can
be targeted to the lysosomes).
Pickart : Would like to determine if polyubiquitin is on htt or some other protein in the
inclusions. What is the half-life of Htt? Is the presence of expanded polygln repeats
toxic to proteasomes in in vitro purified system? Do affected cells have a deficiency
in proteasome activity?
Goldberg : Quoted Tanaka's finding that the half life of the proteasome itself is
approximately 1 week but some subunits may turn over rapidly.
Cummings : Cited data in the literature showing that LMP2-GFP (an interferon
inducible subunit of the proteasome) is translocated into nuclei but only gets out
when the nuclear membrane is destroyed during mitosis.
Baumeister : Proposed using atomic force microscopy for studying inclusions. This
technique can be used with cells and the growth of fibrils can be monitored. Can
also study molecular architecture by optical sections and using EM.
Schwartz : Would obtain brain slices from Bates mice at various times and would look
at frequency of inclusions as marker. He suggested monitoring neuronal
activity/circuitry and looking for a trend in some function : maybe response of
CREB to application of neurotransmitter?
Cummings : Would like to do electrophysiology, especially before inclusions appear.
He would like to monitor changes in gene expression, again before inclusions have
appeared. Also would like the question of whether chaperones are involved in
refolding or directing proteins to the proteasomes?
Gorbea : He would like to have the fates of normal and mutant htt determined. Is
mutant Htt simply in the wrong place by being nuclear? Like Pickart, he wanted to
determine the competency of proteasomes in HD.
Goldberg : Expanded on the theme of brain tissue specificity, this time in connection
with the heat shock response. Mentioned that motor neurons may not have strong
response. Also, aged cells have trouble with the heat shock response to HSF-1.
Would this be related related to age of onset of HD? Also proposed studying the
roles of different chaperones in vitro. Since they may dissociate readily in vivo
Hartl : Proposed studies on Htt biogenesis. Is the newly synthesized protein
associated with chaperones? In vitro, do chaperones modulate aggregation of
polygln expansion repeat proteins? Can the results be extended to cells in culture?
Comments: 1. Bag-1(a SUMO-1-like protein but with no gly-gly repeat) interacts with N-
terminus of Hsp70. It is required for anti-apoptotic activity of Bcl-2 (mechanism is
unknown). 2. Hop brings Hsp70 C-terminus and Hsp90 N-terminus together.
Wilkinson : As described above, he has nuclear & cytoplasmic reporters designed to
assess proteasome activity. Would look at the degradation of polygln repeat
expansion proteins in these cell lines. Also asked if the psi factor in yeast a good
Hochstrasser : Advocated using flies as the model system. Would polygln substitute
for the N-terminus of the psi factor of yeast? Use the known set of mutants in heat
shock genes and proteasome pathway in yeast and study the the same set of genes
Davies : Would like to map the intracellular changes that precede inclusions. Would
also like to know as to what is the basis of the 19S heterogeneity among the
inclusions? (in mouse so far hasn't got the 19S Ab to work in HD sections). If there
is no extracellular staining, then after the cell dies where do the inclusions go?
Goldberg : How come some inclusions don't stain positively for ubiquitin?
Could they have been ubiquitinated initially and ubiquitin removed later? Why isn't
ubiquitin removed from the ones that do stain?
Hochstrasser : Yeast has 17 deubiquitinating enzymes. Are they
compartmentalized/sequestered from the inclusion bodies that stain
immunopositively for polyubiquitin?