Note: Descriptions are shown in the official language in which they were submitted.
WO92/11026 PCT/US91/~9650
-- 1 -- ,
2098922
NEURONAL CHOLINERGIC DIFFERENTIATION FACTOR
1. INTRODUCTION
The present invention relates to a target-
derived neuronal cholinergic differentiation factor
(NCDF), and the therapeutic and diagnostic uses
thereof. The inventio~ provides NCDF, and derivatives,
analogs, and fragments thereof, pharmaceutical
10 compositions of the foregoing, as well as anti-NCDF
antibodies.
2. BACKGROUND OF THE INVENTION
Most sympathetic neurons are noradrenergic;
however, a minority population, including neurons that
innervate sweat glands, are cholinergic. The
sympathetic neurons that innervate sweat glands are
further distinguished in that they contain vasoactive
intestinal pept~de (VIP) and calcitonin gene-related
20 peptide (CGRP) immunoreactivity (Lundberg et al., 1979,
Neuroscience 4, 1539-1559; Landis and Fredieu, 1986,
Brain Res. 377, 177-181; Lindh et al., 1989, Cell
Tissue Res. 256, 259-273), whereas many noradrenergic
neurons conta~n neuropeptide Y (NPY) (Lundberg et al.,
25 1982, Acta. Physiol. Scand. 116, 477-480; Lundberg et
al., 1983, Neurosci. Lett. 42, 167-172; Jarvi et al.,
1986, Neurosci. Lett. 67, 223-227). Although the
mature sweat gland innervation is functionally
cholinergic, the developing innervation is
30 noradrenergic (Landis and Keefe, 1983, Dev. Biol. 98,
349-372; Leblanc and Landis, 1986, J. Neurosci. 6, 260-
265; Stevens and Landis, 1987, Dev. Biol. 123, 179-190;
Landis et al., 1988, Dev Biol. 126, 129-140). When
sympathetic axons first innervate the developing sweat ~
35 glands, they possess intense catecholamine -
histofluorescence and immunoreactivity for the
catecholamine synthetic enzymes, tyrosine hydroxylase
.
. .
WO92/11026 PCT/US91/09650
2098922 - 2 - _~
, ., . ' '
and dopamine ~ hydroxylase. As the gland innervation
matures, catecholamine histofluorescence disappears,
tyrosine and dopamine ~ hydroxylase immunoreactivities
decrease, and cholinergic and peptidergic properties
5 appear. For example, acetylcholinesterase is
detectable at postnatal day 7 (P7), VIP
immunoreactivity at Plo, choline acetyltranferase
activity at P11, and cholinergic transmission at P14.
Thus, the cholinergic sympathetic innervation of sweat
10 glands undergoes a striking change in neurotransmitter
properties during postnatal development.
Several lines of evidence indicate that the
change from noradrenergic to cholinergic function in ~ -
the developing sweat gland innervation is mediated by
interactions with the target tissue. First, when the
innervation of developing sweat glands is delayed by 7- :~
10 days, there i5 a corresponding delay in the
disappearance of catecholamine histofluorescence and
the appearance of cholinergic properties (Stevens and
20 Landis, 1988, Dev. Biol. 130, 703-720). Second, if the
superior cervical ganglion, which contains
noradrenergic sympathetic neurons, is transplanted to
the anterior chamber of the eye with footpad tissue
containing swe~t glands, the neurons innervate the
25 glands, reduce their expression of catecholamine
histofluorescence and NPY, and develop immunoreactivity
for choline acetyltransferase and VIP (Stevens and
s Landis, 1990, Dev. Biol. 137, 109-124). Finally,
cross-innervation experiments provide dir~ct evidence
30 for a target role. When footpad skin is transplanted
,~ ... .
in place of hairy skin in the thoracic region of early ~
postnatal rats, the transplant is innervated by '
sympathetic neurons whose normal targets are
, piloerectors and blood vessels. The sympathetic fibers
~-~ 35 that innervate hairy sXin are noradrenergic and do not ~ -
,
.1
-'~ .
:', , .
:
W092/11026 PCT/US91/09650 ~
~ 3 ~ 20~8922
normally contain choline acetyltransferase activity,
acetylcholinesterase staining, or VIP immunoreactivity
(Schotzinger and L~ndis, 1990, Cell Tissue Res. 260,
S75-S871. Several weeks after innervating the
5 transplanted sweat glands, however, the fibers show a
marked reduction in catecholamine fluorescence and
express properties characteristic of the innervation of
their ~ovel target: they exhibit choline
acetyltransferase activity, acetylcholinesterase
10 staining and VIP immunoreactivity (Schotzinger and
Landis, 1988, Nature 335, 637-639; and unpublished
data). Conversely, if parotid gland, a target of
noradrenergic sympathetic neurons, is transplanted to
the footpad in place of the sweat glands, it is
15 innervated predominantly by fibers that normally
innervate sweat glands and become cholinergic; in this
case, the fibers innervating the transplanted parotid
fall to acquire cholinergic properties and continue to
express catecholaminergic properties typical of the
20 sympathetic innervation of the parotid glands
~Schotzinger and Landis, 1990, Neuron 4, 91-100).
Thus, the normal expression of cholinergic properties
in the sweat gland innervation depends on the presence
of this particular target, and sweat glands are able to
25 induce cholinergic and certain peptidergic properties
in sympathetic neurons that would not normally express
them. Since sympathetic axons never contact sweat
gland cells or the basal lamina that surr,ounds them
directly (Landis and Keefe, 1983, Dev. Biol. 98, 349-
30 372; Uno and Montagna, 1975, Cell Tissue Res. 158, 1-
13; Quick et al., 1984, Anat. Rec. 208, 491-499), it
seems likely that the target effect is mediated by a
soluble factor.
Several proteins that cause a similar
~5 noradrenergic to cholinergic switch in sympathetic
.! , .. . . ,, ' ' , . . . '
' ~ ' ' ' '' i....... ' . , . . . ' ' , , ' ' ,
'' ` ' ' ': . ~. ` ~ . '. ` `. .' ' ' . ', . '. . ' '
WO92~11026 PCT/US91/09650
2~98922 - 4 ~
, . . -
neurons developing in cell culture have been
identified. These represent potential candidates for
the differentiation signal produced by sweat glands.
The cholinergic differentiation factor (CDF) purified
5 from heart cell conditioned medium lPatterson and Chun,
1977, Dev. Biol. 56, 263-280; Fukada, 1985, Proc. Natl.
Acad. Sci. USA 82, 8795-8799) has been shown to be
identical to leukemia inhibitory factor (LIF) (Yamamori
et al., 1989, Science 246, 1412-1416). Ciliary
neurotrophic factor (CNTF), originally identified as a
survival factor for ciliary neurons tAdler et al.,
1979, Science 204, 1434-1436; Barbin et al., 1984, J.
Neurochem. 43, 1468-1478; Manthorpe et al., 1986, Brain
Res. 367, 282-286), and recently cloned (Lin et al.,
15 1989, Science 246, 1023-1025; Stockli et al., 1989,
Nature 342, 920-923), induces cholinergic and reduces
catecholaminergic function in cultured sympathetic
neurons (Saadat et al., 1989, J. Cell Biol. 108, 1807-
1816). Compar~son of the deduced amino acid sequences
20 tyamamori et al., 1989, Science 246, 1412-1416; Stockli
et al., 1989, Nature 342, 920-923) and of the
biological and immunological properties of CDF/LIF and
CNTF ~Rao et al., 1990, Dev. 3iol. 139, 65-74)
~ndicates tha~ they are distinct factors. In addition, ~;
25 a soluble 50 kd cholinergic factor has been obtained
from brain by heparin affinity chromatography (Kessler
et al., 1986, Proc. Natl. Acad. Sci. USA 83, 3528-
3532), and a membrane-associated neurotransmitter-
stimulating factor (MANS) has been solubi~ized and ~ -
, 30 partially p~rified from rat spinal cord. The latter
; activity is associated with a 29 kd protein lWong and
Kessler, 1987, Proc. Natl. Acad. Sci. USA 84, 8726-
8729; Adler et al., 1989, Proc. Natl. Acad. Sci. USA
86, 1080-1083). It is as yet unclear, however, whether
35 the cholinergic-inducing ability of these factors
.
,~
,; :, . , . . . , . , .:, ,, ,., , . ~ . .. - . ... ; , : . - . .,
W092/11026 PCT/US91/09650
20g8922
represents their primary, or even a relevant, function
in normal development. Several of these factors have
been shown in cell culture systems to have additional
functions. For example, CDF/LIF inhibits proliferation
5 and induces macrophage differentiation in the Ml
myeloid cell line (Hilton et al., 1988, Anal. Biochem.
173, 359-367) and maintains the developmental potential
of embyronic stem cells (Smith et al., 1988, Nature
336, 688-690; Williams et al., 1988, Nature 336, 684-
10 687); CNTF has trophic activity for ciliary neurons
(Barbin et al., 1984, J. Neurochem. 43, 1468-1478;
Manthorpe et al., 1986, Brain Res. 367, 282-286) and
induces astrocytic properties in 0-2A progenitor cells
(Hughes et al., 1988, Nature 335, 50-73).
Studies of cholinergic induction in cultured
sympathetic neurons by heart and skeletal muscle cell
' conditioned medium have shown that neurons, once
induced to acquire cholinergic function, maintain it
for a period even if the inducing factor is removed
; 20 from the culture med~um (Patterson and Chun 1977, Dev.
Biol. 56, 263-280; Vidal et al., 1987, Development 101,
617-625). Peptidergic induction is observed in
cultured sympathetic neurons with CDF/LIF (Nawa et al.,
1990, Neuron i, 269-277); withdrawal of CDF/LIF results
25 in the return of substance P content to control levels.
Cross-innervation experiments involving adult
sensory nerves have shown that peptide phenotype can be
altered (McMahon and Gibson, 1987, Neurosci. Lett. 73,
9-15). When a muscle nerve is cross-anastomosed to a
30 cutaneous nerve and induced to innervate targets in the
skin, the regenerated muscle nerves appear to acquire
immunoreactivity for substance P, and conversely, when
-~ the cutaneous nerve was cross-anastomosed to a muscle
nerve, substance P immunoreactivity is decreased in the
i 35 cutaneous nerve.
, . ''' ~. ' '
.
,.
:`
'`:' '.- '' ' . ' : ~ `.' ' . , ' ' ' ' " . : , ,
.
WO92/11026 PCT/~S91/0965~0
2098922 - 6 -
.
3. SUMMARY OF THE INVENTION
The present invention is directed to a
target-derived neuronal cholinergic differentiation
factor (NCDF), and the therapeutic and diagnostic uses
5 thereof. The invention provides NCDF, and derivatives,
analogs, and fragments thereof, pharmaceutical
compositions containing the foregoing, as well as anti-
NCDF antibodies.
The NCDF of the invention is a protein
10 present in extracts of mammalian sweat glands, which
exhibits heat and trypsin lability, lack of substantial
binding to a heparin-agarose affinity column, an
isoelectric point (pI) in the range of approximately
4.8 to 5.2, a non-membrane cellular localization, and
an approximate molecular weight in the range of 16 to
32 kilodaltons. ~he NCDF protein, its derivatives,
analogs, and fragments are able to reduce the
expression of tyrosine hydroxylase and of total
catecholamines, and increase the expression of choline
20 acetyltransferase and vasoactive intestinal peptide
~VIP), by sympathetic neurons in cell culture ( n
vitro).
The NCDF protein, its derivatives, analogs,
and fragments; can be used to induce cholinergic
25 activity in neurons. Such proteins, derivatives,
analogs and fragments can be administered
therapeutically to patients with nervous system damage
or diseases where it is desirable to support survival
and/or cholinergic differentiation of a number of -
30 neuronal types.
' :' '
4. DESCRIPTION OF THE FIGURES
Figure l. Soluble protein extracted from
sweat glands, hairy skin, parotid gland, liver, or
35 sciatic nerve of adult rats was added to cultures of
. ~ , " , . . . . . . .. .. .. . . .. . .. . .
' ' , ' '- '~ . ,' ' . '! . I
.. , . .. . ' ,, , ,,. -,.. .. ...
WO92tl1026 PCT/US91/09650
- 7 - ~
2098922
dissociated sympathetic neurons. Seven days after the
addition of extracts, neurons were homogenized and
aliquots were assayed for levels of choline
acetyltransferase (ChAT) activity by the method of
5 Fonnum (1969, Biochem. J. 115, 465-472). Samples were
run in triplicate. In (a), 250 ~g of protein extracted
from the indicated tissues was added. The data are
expressed as the fold induction of ChAT activity
compared with that present in control cultures grown
10 without added extract, in (b) 250 ~g of protein
extracted from sciatic nerve or sweat gland was added.
The data are expressed as the fold induction of
specific activity per mg of protein added.
Figure 2.
(a) Increasing concentrations of sweat gland
extracts cause increased induction of choline
, acetyltransferase activity. Increasing concentrations ~-~
', of soluble protein extracted from sweat glands of adult
rats were added to sympathetic neuron cultures. Seven
20 days after the addition of extract, neurons were
homogenized and aliquots were assayed for choline
acetyltransferase activity by the method of Fonnum ~-
(1969, ~iochem. J., 115, 465-472). Samples were run in
triplicate. Data are expre~sed as pmol o~ activity per
j 25 min per well + SD.
(b) Time course of induction of choline
acetyltransferase activity. Soluble protein (lOo
~g/ml) extracted from adult sweat glands was added to
sympathetic neuron cultures. Duplicate samples were
~, 30 homogenized at appropriate intervals after the addition
of extract and assayed for choline acetyltransferase
activity. Data are expressed as pmol of activity per
min per well + SD.-
~ Figure 3. Sweat gland extracts reduce
.~ ~ 35 tyrosine hydroxylase. Sympathetic neurons were grown
.. '' ~, ..
`;~
WO92/11026 PCT/US91/Og650
2098922 - 8 -
in medium withOut sweat gland extract (a) or with l00
~g/ml sweat gland extract (b). Samples were pooled
from several wells, homogenized in sample buffer,
electrophoresed, and blotted onto nitrocellulose. The
5 blots were probed with a monoclonal antibody to
tyrosine hydroxylase (inset). The laser densitometer
scan (absorbance of 600 nm) of the staining intensity
of the bands from control and treated cultures is -
shown.
Figure 4.
(a) Sweat gland extracts modulate the
expression of VIP. Serial dilutions of the soluble ,
protein extracted from adult rat sweat glands were
added to sympathetic neuron cultures. Cultures were
15 harvested on the eighth day after the addition of
extract. Sister wells were assayed either for VIP
levels using radioimmunoassay or for choline
acetyltransferase activity. All samples were run in
duplicate. The data are expressed as pg of VIP per
20 well i SD or as pmol of choline acatyltransferase
aativity per well I SD.
~ b) Sweat gland extracts reduce the levels
of NPY and elevate the levels of VIP. Sweat gland
extracts ~l00 ~g/ml) were add~d to sympathetic neuron
25 cultures. Cultures were harvested on the eighth day
after the addition of extract. Sister wells were
assayed for VIP or for NPY by radioimmunoassay. All
samples were run in triplicate. Data are expressed as
pg of VIP or NPY per well + SD.
Figure 5. Appearance of cholinergic
differentiation activity in sweat gland extracts.
Sweat gland extracts were prepared from animals at the
indicated ages. Approximately equal protein
concentrations (lOO ~g/ml) were added to sympathetic
35 neuron cultures. Seven days after the addition of
: . . . . . - .. ... , , . , ~ ~
. :. , ~ - : . , . . :
,,
WO92/11026 PCT/US91/09650
~ 9 20sss22~ ,
extract, neurons were harvested, and aliquots were
assayed for choline acetyltransferase activity. At
least three different preparations at each age were
tested. Data are expressed as fold induction of
5 choline acetyltransferase per mg of extract protein +
SD.
Figure 6. The cholinergic-inducing activity
present in sweat gland extracts is not
immunoprecipitated with antibodies to CDF/LIF.
(a) Sweat gland extracts (DEAE fraction)
were incubated with protein A-Sepharose (A), affinity-
purified antibodies to the N-terminal sequence of CDF
(B), or affinity-purified antibodies preincubated with
the peptide antigen (C). After immunoprecipitation,
15 supernatants were added to sympathetic neuron cultures.
Ten days after the addition of extract, the cultures
were assayed for choline acetyltransferase activi~y by
the method of Fonnum ~1969, Biochem. J., 115, 465-472). ;
~he results are expressed as the fold induction of -
20 choline acetyltransferase compared with the activity
observed in neurons grown in medium without extract.
All samples were run in duplicate.
(b) ~25I-labeled recombinant CDF/LIF ~20,000
cpm) was incu~ated with aff'in~ty-purified antibodies to
25 the N-terminal sequence o~ CDF ~lanes 1 and 3) or
affinity-purified antibodies preincubated with the
peptide antigen (lanes 2 and 4) in buffer (lanes 1 and
2) or with 10 ~g of soluble protein extracted from -
adult rat sweat gland (lanes 3 and 4). Following
30 immunoprecipitation by affinity purified antibodies to
the N-terminal sequence of CDF, the labeled proteins
were extracted by boiling in SDS sample buffer and
subjected to SDS-PAGE in a 10% gel. The labeled LIF
(arrow) was localized on X-ray films developed after a
35 7 day exposure. The higher molecular weight band is
:. '
,
:'~:.''-' " ' ' ,: - .~; ': ' ' ' ',, ' ' ', ' . ,:' ,.'' . ' ; . ,' . ,, :, . .
WO92t11026 PCT/US91/09650
2098922 - lO
,',..'.:'.
labeled bovine serum albumin immunoprecipitated by
protein A-Sepharose.
FIG. 7. CNTF is not detectable in sweat ) -
gland extracts. In panel a, lO ng of recombinant CNTF
5 was blotted onto nitrocellulose. In panels b and c, 60 ,
~g of soluble protein (DEAE fractions) from sciatic
nerve extract (lane l), from hairy sXin extract of -
adult rat (lane 2) or from sweat gland extract of adult
(lane 3) or 21 day (lane 4~ animals (panels b and c)
were blotted onto nitrocellulose. In panels a and b,
the blots were probed with a polyclonal antiserum
raised against recombinant rat CNTF, while in panel c
the blot was probed with antibody preincubated with lO ~;
~m recombinant CNTF. Panel a documents that the
15 antiserum recognizes CNTF (arrowhead). As expected,
the antiserum recognizes a 24 kilodalton (kd) band
, present in sciatic nerve extracts (lane l b,c), but no
specific bands were evident in hairy skin extracts -
~lane 2) or ln sweat gland extracts from 21 day (lane
20 3) or adult ~lane 4) animals. Arrowheads in b and c
~ indicate 92, 30 and 22.5 kd standards.
¦ Figure 8. CNTF message is not detectable in
:J sweat gland extracts. 30 ~g of total RNA from adult
sciatic nerve ~a), sweat gland ~b), liver ~c) and optlc
25 nerve ~d) was electrophoresed and transferred onto
nylon membrane. The membrane was then probed with an
oligonucleotide probe to rat CNTF. Arrow shows a
, positive l.3 kb band in lane a containing sciatic nerve
RNA and a fainter band in the same positi6n in optic
30 nerve ~d). No specific signal is detected in l~nes b
and c containing sweat gland and liver RNA,
' respectively.
Figure 9. In Situ Hybridization. Sections
of sciatic nerve were probed with an oligonucleotide
35 probe to rat CNTF. Panel a shows specific
.j:. .
-: .
.. .
' : -
... .. : . ~. ~. - . -. - - .. . , . . . . -
: .. i , . ~ . . , , , . ,, . : ,
WO92J11026 PCT/US91/09650
-- 11 --
2098922
hybridization to Schwann cells in sciatic nerve
sections (with an antisense probe). Panel b shows the -
same tissue section stained with ethidium bromide. No
~inding is seen with the sense (control) probe in Panel
5 c, which shows a random distribution of grains. Panel
d shows the same tissue section stained with ethidium
bromide.
Figure lO. In Situ Hybridization. Sections
of sweat gland were probed with an oligonucleotide
10 probe to rat CNTF, as used in Fig. 9. No specific
binding is seen in sections of sweat glands (Panel a).
No binding is seen with the sense (control) probe
~Panel c). Panels b and d represent ethidium bromide
stained sections.
Figure ll. Anion exchange chromatography.
After homogenization and centrifugation as described in
Section 6.3.3., the sweat gland extract supernatant was
applied to a DEAE ion exchange column, and assayed for
choline acetyltrans$erase ~ChAT) inducing activity in
20 sympathetic neurons. Closed squares: ChAT induction.
Closed diamonds: NaCl gradient.
Figure 12. Chromatofocussing. The DEAE
eluate was chromatographed on a MONO P column, and 0.5
ml extracts we're collected and assayed for cholinergic
25 activity ~ChAT inducing activity in sympathetic
neurons). Closed squares: ChAT induction. Closed
diamonds: pH. - -
Figure 13. (a) SDS gel fractions betwen 22- ~
26 kd and 26-32 kd were eluted and added to cultures of -
30 dissociated sympathetic neurons. Seven days after the
addition of extracts, neurons were homogenized and
aliquots were assayed for levels of choline
acetyltransferase (ChAT) activity by the method of
Fonnum. Samples were run in triplicate. The data are
35 expressed as the fold induction of ChAT activity -~
::
: . ~. . - , . , .. - ... - , , , ~ ~ : ...... .
W O 92~11026 PCT/US91/09650 ,
2098922 - 12 -
compared to that present in control cultures grown
without added extract.
In b, aliquots of the eluted protein were
rerun on an SDS gel and stained with Coomassie blue.
5 Lane a shows the 22-26 kd (lower arrow) fraction and
lane b the 26-32 (upper arrow) kd fraction.
, : '
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention i9 directed to a
10 target-derived neuronal cholinergic differentiation
factor (NCDF), and the therapeutic and diagnostic uses
thereof. The invention provides NCDF, and derivatives,
analogs, and fragments thereof, pharmaceutical
compositions containing the foregoing, as well as anti-
15 NCDF antibodies.
The NCDF of the invention is a proteinpresent in extracts of mammalian sweat glands, which
exhibits heat and trypsin lability, lack of substantial
binding to a heparin-agarose a~finity column, an
i 20 isoelectric point (pI) in the range of approximately
4.8 to 5.2, a non-membrane cellular localization, and
an approximate molecular weight in the range of 16 to
32 kilodalton5. The NCDF protein, its derivatives,
analogs, and ~ragments are able to reduce the
25 expression of tyrosine hydroxylase and of total
catecholamines, and increase the expression of choline
acetyltransferase and vasoactive intestinal peptide
(VIP), by sympathetic neurons in cell culture (in
vitro)
The NCDF protein, its derivatives, analogs,
and fragments, can be used to induce cholinergic
activity in neurons. Such proteins, derivatives,
analogs and fragments can be administered -
therapeutically to patients with nervous system damage ~ -
, 35 or diseases where it is desira~le to support survival
.` : '
.
., .
WO92/11026 PCT/US91/09650
2098922
and/or cholinergic differentiation of a number of
neuronal types. -
In a specific embodiment of the invention,
the NCDF protein is that found in extracts of human
5 sweat glands. In another embodiment, the NCDF protein
is that found in extracts of sweat glands from rats.
In a further embodiment of the invention, the
NCDF protein, its derivatives, analogs, and fragments
may induce the expression of additional peptides such
10 as enkephalin, somatostatin, and substance P.
As detailed in the examples sections, infra,
we assayed the effects of sweat gland extracts on the
transmitter properties of cultured sympathetic neurons.
We found that there exists a soluble factor present in
sweat glands (which we term NCDF), which reduces the
expression of catecholamines and tyrosine hydroxylase
and induces the expression of choline acetyltransferase
and VIP.
,
5.l. THE NCDF PROTEIN, DERIVATIVES,
ANALOGS AND F~GM~NTS
Human, rat, porcine and other species NCDF,
or their functional equivalents, can be used in
accordance wi~h the invention. Additionally, the
25 invention relates to NCDF proteins isolated ~rom ovine,
bovine, ~eline, avian, equine, or canine, as well as
primate sources and any other species in which NCDF
activity exists. The invention also provides for NCDF
proteins, fragments and derivatives thereof or their
30 functional equivalents. The invention also provides
fragments or derivatives of NCDF proteins which -;
comprise antigenic determinant(s) or which are
functionally active. As used herein, functionally
active shall mean having positive activity in assays
35 for known NCDF function, e.g. the ability to increase ~-
..'.
.-
' " .:
,, ~ : . ,' . :
',: . , : . : ' ' ' : , ', ' , , ' ' . ''
-~
'. ' ' . '' :
WO92/11026 - 14 - PCT/US91/09650
2098922
the expression of choline acetyltransferase by
sympathetic neurons ln vitro.
The NCDF derivatives, analogs, or fragments
of the invention include, but are not limited to, those
5 containing all or part of the primary amino acid
sequence contained in the full-length NCDF protein as
purified from sweat gland extracts, including altered
sequences in which functionally equivalent amino acid
resldues are substituted for residues within the
10 sequence resulting in a silent change. For example,
one or more amino acid residues within the sequence can
be ~ubstituted by another amino acid of a similar
polarity which acts as a functional equivalent,
resulting in a silent alteration. Substitutes for an
ami~o acid within the sequence may be selected from
other members ~f the class to which the amino acid
belongs. For example, the nonpolar (hydrophobic) amino
acids include alanine, leucine, isoleucine, valine,
proline, phenylalanine, tryptophan and methionine. The
20 polar neutral amino acids include glycine, serine,
threonine, cysteine, tyrosine, asparagine, and
glutamine. The positively charged ~basic) amino acids
include arginine, lysine and histidine. The negatively
charged ~acidic) amino acids include aspartic acid and
25 glutamic acid. Also included within the scope of the
invention are NCDF proteins, fragments, analogs or
derivatives thereof which are modified, e.g., by
proteolytic cleavage, linkage to an antibody molecule
or other cellular ligand, acetylation, formylation,
30 oxidation, reduction, etc.
5.2. PURIFICATION OF NEURONAL CHOLINERGIC
DIFFERENTIATION FACTOR
NCDF may be purified from any available
source of mammalian sweat glands using techniques known -
:- : :- , ~ - .
. ~ - . .
WO92/11026 PCT/US91/09650
- 15 -
2 0 9 g ~ 2 2
in the art. Such techniques include but are not
limited to chromatography (e.q., ion exchange,
affinity, and sizing column chromatography),
centrifugation, differential solubility, or by any
5 other standard technique for the purification of
proteins.
For example, and not by way of limitation, it
is envisioned that NCDF may be isolated from sweat
gland extracts according to the following method.
10 Sweat gland extracts may be prepared according to the
method set forth in Section 6.3.3. After
homogenization and centrifugation as set forth therein,
the supernatant may be collected and applied to an
anion exchange column (e.g. DEAE, Whatman DE52
cellulose equilibrated in phosphate buffer), and
collected therefrom by methods known in the art.
Purified extract may then be subjected to sucrose
; gradient centrifugation by known methods, with the
; appropriate fraction concentrated by ultra filtration.
20 The purified NCDF may then be subjected to analytic or
preparative polyacrylamide gel electrophoresis. If
desired, following elution from a polyacrylamide gel,
NCDF may be further purified and freed from certain
bu~fer compon~nts by use of a HPLC reverse phase
25 column.
As an example of gel electrophoresis,
purified NCDF may be analyzed using a slab SDS-
polyacrylamide gel. Purified NCDF or molecular weight
standards may be electrophoresed and the gel cut out
30 and processed as follows: the polypeptides may be
visualized without fixation by precipitating the
protein-associated SDS during an incubation of the gel
` in o.Z5 ~ KCl and recording the positions of the
standards and NCDF bands. Lanes may then be fixed and
35 stained with Coomassie blue. Other lanes may then be
., ' ' ~ .
.: .
,
.
~....... , , . .... ~ . . -. ~ .
`:: .'. . - ' ' . , ' . - , . . :
.', '. ' - ' :. . . - , ..
W O 92/t1026 P ~ tUS91/09650
~09~922 - 16 -
cut into slices, and eluted, e.g. by electroelution or
by incubation with Triton X-100, and then the eluates
may be assayed for NCDF activity.
5.3. NCDF BIOASSAYS
NCDF activity may be evaluated using any
NCDF-sensitive in yivo or in vitro systems. For
example, assays including including but not Iimited to
those described in Sect~on 6.3., infra, may be used,
10 e.g., those assaying the ability to increase the
expression of choline acetyltransferase or increase the
expression of vasoactive intestinal peptide, or reduce
the expression of tyrosine hydroxylase, or reduce the
expression of total catecholamines, by sympathetic
15 neurons in cell culture.
Alternatively, as but another example, it is
envisioned that NCDF activity may be measured by
quantitating 24-hour survival of embryonic (E8) chick
ciliary ganglion ~CG) neurons in monolayer cultures.
20 For example, ciliary ganglia may be collected fro~ E8
chick embryos, dissociated (yielding approximately --
20,000 cells per ganglion) and then diluted in HEBM
medium containing 20 percent horse serum as described
-in Varon et ai. ~1979, Brain Res. 173, 29-45). About
25 fifty microliters of cell suspension containing 1,000
neurons ~2,000 cells) may then be seeded into
microtitre dishes and then putative NCDF activity may
be added. Culture plates may then be maintained at
37C in 5% C2 for 24 hours, after which the cultures
30 may be fixed by the addition of 200 ~1 2 per cent -
glutaraldehyde in HEBM medium, and the number of
surviving neurons may be determined visually by direct
- count under phase contrast microscopy.
~5
5.4. SEOUENCING NCDF
WO92/t1026 PCT/US91/09650
2098922
The NCDF protein may be sequenced directly or
initially cleaved by any protease or other compound
known in the art, including, but not limited to,
Staph~lococcus aureus V8, trypsin, and cyanogen
5 bromide. Peptides may be sequenced by automated Edman
degradation on a gas phase microsequencer according to
the method of Hewick et al. (1981, J. Biol. Chem. 256,
7990-7997) and Hunkapiller et al. (1983, Methods
Enzymol. 91, 227-236). Detection of
10 phenylthiohydantoin amino acids may then be performed
according to Lottspeich (1985, Chromatography 326, 321-
327). Overlapping fragments of amino acid sequence may
be determined and used to deduce longer stretches of
co~tiguous sequence.
5.5. GENERATION OF ANTI-NCDF ANTIBODIES
According to the invention, NCDF protein, or
fragments or derivatives thereof, may be used as
immunogen to generate anti-NCDF antibodies.
Various procedures known in the art may be
used ~or the production of polyclonal antibodies to
epitopes of NCDF. For the production of antibody,
various host animals can be immunized by injection with
NCDF protein, or a fragment or derivativs thereof,
25 including but not limited to rabbits, mice, rats, etc. -
Various adjuvants may be used to increase the ;~
immunological response, depending on the host species, -
and including but not limited to Freund's (complete and
incomplete), mineral gels such as aluminum hydroxidé,
30 surface active substances such as lysolecithin,
pluronic polyols, polyanions, peptides, oil emulsions, -
keyhole limpet hemocyanins, dinitrophenol, and
potentially useful human adjuvants such as BCG tBacille
Calmette-Guerin) and, Corynebacterium parvum. -
; ,~
..
,'' ' . . ,'.' ' . ' . ' ' ' '. ' : ' " ' '. ' " ; . ~ '. : . '' ": '
., . -:, . ' ' ' . . i , ,. , , ` ,, ., : ,
WO92/11026 PCT/US91/09650
- 18 -
2098922
If desired, to further improve the likelihood
of producing an anti-NCDF immune response, once
obtained, the amino acid sequence of NCDF may be
analyzed in order to identify portions of the molecule
`5 which may be associated with increased immunogenicity.
For example, the amino acid sequence may be subjected
to computer analysis to identify surface epitopes,
according to the method of Hopp and Woods (1981, Proc.
Natl. Acad. Sci. U.S.A. 78, 3824-3828).
For preparation of monoclonal antibodies
directed toward NCDF, any technique which provides for
the production of antibody molecules by continuous cell
lines in culture may be used. For example, the
hybridoma technique originally developed by Kohler and
15 Milstein (1975, Nature 256, 495-497), as well as the
i, trioma technique, the human B-cell hybridoma technique
(Kozbor et al., 1983, Immunology Today 4, 72j, and the
EBV-hybridoma technique to produce human monoclonal
antibodies (Cole et al., 1985, in "Monoclonal
20 Antibodies and Cancer Therapy," Alan R. Liss, Inc. pp.
77-96) and the like are within the scope of the present
.; ..
; invention.
The monoclonal antibodies for therapeutic use
~- may be human monoclonal ant~bodies or chimeric human-
~ 25 mouse (or other species) monoclonal antibodies. Human
1 monoclonal antibodies may be made by any of numerous
techniques known in the art (e.a., Teng et al., 1983,
Proc. Natl. Acad. Sci. U.S.A. 80, 7308-7312; Kozbor et
`al., 1983, Immunology Today 4, 72-79; Olsson et al.,
30 1982, Meth. Enzymol. 92, 3-16). Chimeric antibody
molecules may be prepared containing a mouse antigen-
binding domain with human constant regions (Morrison et
al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81, 6851,
Takeda et al., 1985, Nature 314, 4S2.
" 35
. ~ .
, .
' '' ' '' , '"~ ' ', ''' . ' ' . , , ~ '. ,
" i , , . . " . ,, , ,. . ` , , : ', " ' , ~, . ,, . ,, . " , ,. ' ~ . " ' ", . " ' : ,
W092/11026 PCT/US91/09650
-- 19 --
~x~ ~ 9~
A moiecular clone of an antibody to a NCDF
epitope can be prepared by known techniques.
Recombinant DNA methodology (see e.g., Maniatis et al.,
1982, Molecular Cloning, A Laboratory Manual, Cold
5 Spring Harbor Laboratory, Cold Spring Harbor, New York)
may be used to co~struct nucleic acid sequences which
encode a monoclonal antibody molecule, or antigen
binding region thereof.
Antibody molecules may be purified by known j!~
10 techniques, e.a., immunoabsorption or immunoaffinity
chromatography, chromatographic methods such as HPLC
(high performance liquid chromatography), or a
combination thereof, etc.
Antibody fragments which contain the idiotype
of the molecule can be generated by known techniques.
Fox example, such fragments include but are not limited
to: the F~ab' )2 fragment which can be produced by -
pepsin digestion of the antibody molecule; the Fab'
~ragments which can be generated by reducing the
20 disulfide bridges of the F(ab' )2 fragment, and the 2 Fab
or Fab fragments which can be generated by treating the
antibody molecule with papain and a reducing agent.
5.6. UTIL~Y OF THE INVENTION
The present invention relates to NCDF and to
peptide fragments, analogs, or derivatives produced
therefrom. NCDF protein, peptides, and derivatives, -
and anti-NCDF antibodies, may be utilized in diagnostic -
and therapeutic applications. For most purposes, it is
30 preferable to use NCDF from the same species for
diagnostic or therapeutic purposes, although cross- 7
species utility or NCDF may be useful in specific
embodiments of the invention.
. .
. :'
: '
.. .. .. ~ .. , . . . . . .. . ..... ~.. . ... , .. , .. ,. , .. . . . ,"........ .
: :.: .. : : .. . . - .: ,.. . . . - .: .. : . : ,, , - .. : .: ., . . , ... , . . : .
WO92/11026 PCT/US91/09~50 :
- 20 -
20--9~922 ~ -:
5.6.1. DIAGNOSTIC APPLICATIONS
The present invention, which relates to NCDF :
protein, peptide fragments, or analogs or derivatives
produced therefrom, as well as antibodies directed
5 against NCDF protein, peptides, or derivatives, may be
` utilized to diagnose diseases and disorders of the
; nervous system which may be associated with alterations
in the pattern of NCDF expression.
Assays can be used to detect, prognose,
10 di~gnose, or monitor cond~tions, disorders, or disease
states associated with changes in NCDF expression,
including, in particular, conditions resulting in
damage and degeneration of neurons which may respond to
NCDF, such as parasympathetic neurons, cholinergic
15 neurons, spinal cord neurons, neuroblastoma cells and
cells of the adrenal medulla. Such diseases and
conditions may include but are not limited to trauma,
infarction, infection, degenerative nerve disease,
malignancy, or post-operative changes including but not
20 limited to Alzheimer's Disease, Parkinson's Disease,
Huntington's Chorea, and amyotrophic lateral sclerosis.
In alternate embodiments of the invention,
~3 antibodies directed toward NCDF protein, peptide
~ ~ragments, anaIogs or derivatives can be used to
.3 25 diagnose disease~ and disorders of the nervous system,
including, in particular, those neuronal populations
and clinical disorders and diseases listed supra.
Antibodies directed toward NCDF proteins of the
invention can be used, for example, in in situ ` -
30 hybridization techniques using tissue samples obtained
from a patient in need of such evaluation. In a
further example, the antibodies of the invention can be
; used in ELISA procedures to detect and/or measure
amounts of NCDF present in tissue or fluid samples;
35 similarly, the antibodies of the invention can be used
'i~ :
,, .
. .
' . , ; . ! , .. . ,, .. ' ~ ,. ~ ' ' i
WO92/11026 PCT/US91/09650
- 21 -
2o98922 ' ' ' '
in Western blot analysis to detect and/or measure NCDF
present in tissue or fluid samples. .
The immunoassays which can be used to detect
or measure NCDF protein, its analogs, derivatives or
5 fragments, include but are not limited to competitive
and non-competitive assay systems using techniques such
as radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays,
precipitin reactions, gel diffusion precipitin
10 reactions, immunodiffusion assays, agglutination .
assays, complement-fixation assays, immunoradiometric
assays, fluorescent immunoassays, protein A
immunoassays, and immunoelectrophoresis assays, to name .
but a few.
In further embodiments of the invention, NCDF
protein, peptide fragments or derivatives can be used . .
to diagnose diseases and disorders of the nervous
system. In a particular embodiment and not by way of :
limitation, labeled NCDF protein or peptide fragments
20 can be used to identify tissues or cells which express
the NCDF receptor in order to identify aberrancies of
NCDF receptor expression and consequently, potential ~:~
abnormalities in the tissue or cellular response to ;
NCDF .
5 . 6 . 2 . T~iERAPEUTIC APPLICATIONS - .::
The present invention, which relates to NCDF :~
protein, peptide fragments, analogs or derivatives
produced therefrom, as well as to antibodies directed
30 against NCDF protein, peptides, analogs or derivatives,
may be utilized to treat diseases and disorders of the
nervous system which may be associated with alterations
~ in the pattern of NCDF expression or which may benefit .
from exposure to NCDF or anti-NCDF antibodies.
::~
'"',.':
.
'.
. A , ., . . ; . . , .. . ~ . . ' ' ~ . .. . .
.~'` . " ' ' . ' ' . . ' ' '' ~. . . ' . . ' ".' ' ' ,'' ' ' . .. . . '.. ' . '. .. '. . ' . . ' . . .' .
WO92/11026 PCT/US91/09650
2~98922 22 -
NCDF, and its derivatives, fragments, and
analogs, can be used to support the survival and
cholinergic differentiation of a number of neuronal
types, including spinal motor neurons, parasympathetic
5 neurons of the ciliary ganglion, etc. The NCDF
products of the present invention may have utility in
supporting ln yivo the survival and differentiation of
certain cell populations, including but not limited to,
spinal motor neurons, parasympathetic neurons
10 (including ciliary ganglion neurons which innervate the
iris, heart, gastrointestinal tract and other visceral
structures). Thus, in specific embodimentq of the
present invention, a pharmaceutical preparation
containing NCDF or its active derivatives, fragments or
15 analogs, can be administered to patients in whom the
central nervous system is damaged. In another
embodiment of the invention, a pharmaceutical
preparation containing NCDF or its active derivatives,
fragments, or analogs, alone or in combination with
20 another neurotrophic factor (e.g. CNTF, NGF, BDNF
(brain-derived neurotrophic factor), or NT-3 -
(neurotrophin-3)) can be administered to patients
suffering from pathological conditions resulting from
damage to or ~ysiological imbalance, overactivity or
25 underactivity of the autonomic nervous system, or
conditions which might be aggravated by such autonomic
nervous system damage or imbalance. Such disorders
might include, but are not limited to: chronic
anhidrosis and hyperhidrosis, cardiac arhythmias,
30 chronic constipation, neurogenic bladder dysfun~tion
and ejaculatory disturbances.
In various embodiments of the invention, NCDF
protein, peptide fragments or derivatives can be
administered to patients in whom the nervous system has -
35 been damaged by trauma, surgery, ischemia, infection
.'
...... , . .. .. .... ..... ~ ........ .. . ..... . ........ ,, . . . , ~ . ..
~ .. ,. . -- - -. - ~ . ,: , . : . : - ....... ...... - : .: :. .
. .. ........ . - .. .... : . :. . - . ., . .. . .. . . .:
W092/l1026 PCT/US91/09650
- 23 - 2~9~22
(e.g. polio or-A.I.D.S.), metabolic disease,
nutritional deficiency, malignancy, or toxic agents.
The invention in particular can be used to treat
conditions in which damage has occurred to neurons, by
5 administering effective therapeutic amounts of NCDF
protein or peptide fragments or derivatives or analogs.
In various specific embodiments of the invention, NCDF
can be administered to spinal cord neurons which have
been damaged, for example, by trauma, infarction,
10 infection, degenerative disease or surgical lesion. It
may be desirable to administer the NCDF-related
peptides or NCDF protein by adsorption onto a membrane,
e.g. a silastic membrane, that could be implanted in
the proximity of the damaged nerve. The present
5 invention can also be used for example in hastening the
recovery of patients suffering from diabetic
neuropathies, e.g. mononeuropathy multiplex or ;`~
r~ impotence. In further embodiments of the invention,
NCDF protein or peptide fragments or derivatives
` 20 derived therefrom, can be used to treat congenital
I condition5 or neurodegenerative disorders, including,
i but not limited to, Alzheimer's disease, ageing,
peripheral neuropathies, ~arkinson's disease,
Huntington's ~orea and diseases and disorders of
25 motorneurons; in particular, the invention can be used
to treat congenital or neurodegenerative disorders
associated with cholinergic neuron dysfunction.
In a specific embodiment of the invention, it
~ is envisioned that administration of NCDF protein, or
r' 30 peptide fragments or derivatives derived therefrom, can
be used in conjunction with surgical implantation of
7 tissue or other sustained release compositions in the
treatment of Alzheimer's disease, amyotrophic lateral
- sclerosis and other motorneuron diseases (including, -
35 for example, Werdnig-Hoffman disease), and Parkinson's ~
.
: . . . . . . . . . .
WO92/11~26 ~i PCT/US91/09650
- 24 -
2098922
disease. NCDF may also be useful in the treatment of a
variety of dementias as well as congenital learning
disorders.
In further embodiments of the invention, NCDF
5 protein, fragments or derivatives can be used in -
conjunction with other cytokines to achieve a desired
neurotrophic effect. For example, and not by way of
limitation, according to the invention NCDF can be used
together with NGF to achieve a stimulatory effect on
10 growth a~d survival of neurons. It is envisioned that
NCDF may function synergistically with other CNS-
derived peptide factors yet to be fully characterized,
in the growth, development, and survival of a wide
array of neuronal subpopulations in the central and
15 peripheral nervous system.
In still further embodiments of the
invent~on, antibodies directed toward NCDF protein, or
peptide fragments or derivatives thereof, can be
administered to patients suffering from a variety of
20 neurologic disorders and diseases and who are in need
of ~uch treatment. For example, patients who suffer
from excessive production of NCDF may be in need of
such treatment. Anti-NCDF antibodies can be used in
prevention o~ a~errant r~generation o~ sensory neurons
25 (e.g. post-operatively), or in the treatment of chronic
pain syndromes.
5.6.3. PHARMACEUTICAL COMPOSITIONS
The active compositions of the invention,
30 which may comprise all or portions of the NCDF protein,
peptide fragments or analogs or derivatives produced -~
therefrom, or antibodies ~or antibody fragments)
directed toward NCDF protein, peptide fragments, or
derivatives, or a combination of NCDF and a second
35 agent ~such as NGF) may be administered in any sterile
:: .: , , ~ ,. , . . : . ., . ,.-~ ~, . .. , . . .. .. : - -
WO92t11026 PCT/US91/09650
- 25 -
'~09~922
biocompatible pharmaceutical carrier, including, but
not limited to, saline, buffered saline, dextrose, and
water.
The amount of NCDF protein, peptide fragment,
5 derivative, or antibody which will be effective in the
treatment of a particular disorder or condition will
depend on the nature of the disorder or condition, and
can be determined by standard clinical techniques.
Where possible, it ls desirable to determine the dose- ;
10 response curve first ~a Yitro, e.g. in the NCDF
bioassay systems described supra, and then in useful
animal model systems prior to testing in humans.
Methods of introduction include but are not
limited to intradermal, intramuscular, intraperitoneal,
15 intravenous, subcutaneous, oral, and intranasal. In
addition, it may be desirable to introduce the
pharmaceutical compositions of the invention into the
central nervous system by any suitable route, including !'.
intraventricular and intrathecal injection;
20 intraventricular in~ection may be facilitated by an
intraventricular catheter, for example, attached to a
reservoir, such as an Ommaya reservoir.
Further, it may be desirable to administer
the pharmaceu ~cal compositions o~ the invention
25 locally to the area in need of treatment; this may be
achieved by, for example, and not by way of limitation, ;
local infusion during surgery, by injection, by means ~ -
of a catheter, or by means of an implant, said implant
being of a porous, non-porous, or gelatinous material, ---
30 including membranes, such as sialastic membranés, or
fibers.
The invention also provides for
pharmaceutical compositions comprising NCDF proteins,
peptide fragments, analogs, or derivatives administered
35 via liposomes, microparticles, or microcapsules. In
,
.
.
WO92/11026 PCT/US91/09650
- 26 -
2098922
various embodiments of the invention, it may be useful
to use such compositions to achieve sustained release
of NCDF and NCDF-related products.
6. CHARACTERIZATION OF A TARGET-DERIVED
NEURONAL CHOLINERGIC DIFFERENTIATION FACTOR
The sympathetic innervation of rat sweat
glands undergoes a target-induced switch from a
noradrenergic to a cholinergic and peptidergic
phenotype during development. Treatment of cultured
sympathetic neurons with sweat gland extracts mimics
many of the charges seen n vivo. Extracts induce
choline acetyltransferase activity and vasoactive
intestinal peptide expression in the neurons in a dose-
15 dependent fashion while reducing catecholaminergic
properties and neuropeptide Y. The cholinergic
differentiation activity appears in developing glands
of postnatal day S rats and is maintained in adult
j glands. It is a heat-labile, trypsin-sensitive, acidic
20 protein that does not bind to heparin-agarose.
Immunoprecipitation experiments with an antiserum
directed against an N-terminal peptide of a cholinergic
differçntiation factor (CDF/LIF) from heart cells
suggest that the 5weat gland differentiation factor is
25 not CDF/LIF. The sweat qland activity is a likely ~-
candidate for mediating the target-directed change in
sympathetic neurotransmitter function observed ~ vivo. -
To identify the target factor responsible for
the adrenergic to cholinergic switch obse~ved in the
30 developing sweat gland innervation and to explore the
possible in vivo role of the candidate cholinergic
factors identified in cell culture, we assayed the
effects of sweat gland extracts on the transmitter
~` properties of cultured sympathetic neurons. We found
that sweat glands contain a soluble factor(s) with the -
appropriate spectrum of activities: it reduces the
' : '.
.~ . .
~. . . - .
' . ! ' , . ' ' ' ' ' . ' ' : : ' ' ' '
` ' . ' ' ' ` ' '. :` ` ' ' .'' ' ' " ""'. " ' ~` : ' '. " ' ' '' ' '.'' ' ' ' ' "' ' ' ', ' ' ` . .' . .` ` ~ ' ,
WO92/11026 PCT/US91/09650
- 27 - '~ 9~2 ~
expression of catecholamines and tyrosine hydroxylase
and induces the expression of choline acetyltransferase `
and VIP. This activity is present when the phenotype
of the sweat gland innervation is changing. Our
5 initial characterization of the sweat gland-derived
choline acetyltransferase-inducing activity permits a
comparison with the cholinergic factors previously
identified in cell culture.
6.l. RESULTS
6.l.l. SWEAT GLAND EXTRACTS INDUCE CHOLINE ACETYL-
TRANSFERASE ACTIVITY IN SYMPATHETIC NEURONS
To examine the effects of soluble factors
present in sweat glands on neurotransmitter status,
extracts from the footpads of adult rats were added to
sympathetic neuron cultures at a concentration of 250
~g of extract protein per ml. Neurons in sister wells
were grown either in medium without added tissue '
extract or in medium containing an equal protein
20 concentration of liver, hairy skin, or parotid gland
extracts prepared in the same manner as the sweat gland -
extracts. The addition of sweat gland extract caused a
15-fold induction in choline acetyltransferase activity :'
compared with ~eurons grown in medium alone or with
25 extracts of liver, hairy skin or parotid gland (Figure
la).
one potential source of the cholinergic-
inducing activity in the sweat gland extracts is the
possible presence of CNTF in the peripheral nerve
30 plexus of the footpad tissue. Comparison of the
cholinergic-inducing activity in sciatic nerve extracts
and sweat gland extracts ~Figure lb), however, -
indicated that the level of induction per mg of extract
protein was similar despite the fact that the
35 peripheral nerve plexus constitutes only a small
proportion of the gland tissue. This observation and
': . . . ' : ~,, . , .~ , I , . .
` '~ ' ' .'' . ' . ' . ' ' ' '' ' -, ',,: ' - , '
.: , ; ,.. , , .. - ~ .... . . . ' . . , . :, ,
: : ' . ' . ' , :, .' ~ '
W O 92/11026 PC~r/US91/09650
2 2 - 28 -
the finding that extracts of hairy skin did not cause
choline acetyltransferase induction in cultured
sympathetic neurons even though the hairy skin contains
a plexus of sympathetic and sensory nerve fibers and
5 endings comparable to that in sweat gland-containing
skin make it unlikely that the ability of sweat gland
extracts to induce cholinergic function is due to CNTF
potentially derived from Schwann cells (Stockli et al.,
1989, Nature 342, 920-923).
; . -
: ' .
.
. .
' 25
'~
.
. .
,
., -,,
, 35 : ~
.:
:, , . . . ' .~; ' ' , ' , .. ,,'., , ' ~ '.'' ' ", , . '. ' , ' " " ', ' ' ' . ' '
WO92/11026 PCT/US91/09650
- 29 ~ 2 098922
TABLE I
The Cholinergic-Inducing Effect of Sweat Gland ;
Extracts Is Independent of Serum
Choline Acetyltransferase
Activity
_ Medium SG Extract
Defined medium 8.02 + 1.64 109.83 +
5.57
L15-C02 + serum 18.05 + S.57 126.06 + .
5.58
Sympathetic neurons were cultured in L15-C02 either '
lacking serum or containing 5% rat serum with 300 ~g/ml
sweat gland extract. Cells were harvested 7 days after
the addition of extracts and aliquots were tested for
choline acetyltransferase activity by the method of
Fo~num ~1969, Biochem. J. 115, 465-472). Samples were
20 run in triplicate. Data are expressed as pmol of
choline acetyltransferase per min per well + SEM.
.
The~ nduction o~ choline aaQtyltransferase
25 activity by the sweat gland extract was due to a direct -
effect on sympathetic neurons. Since the neurons were
grown in the continuous presence of 10 ~M cytosine
arabinoside, nonneuronal cells were virtually absent;
thus, it is unlikely that the sweat gland~extracts
exerted their influence indirectly via nonneuronal
cells. In addition, when sympathetic neuron cultures
were maintained in serum-free medium that yielded
cultures free of nonneuronal cells, cholinergic
induction was seen following treatment with sweat gland
, 35 extracts (Table I). ~This observation also makes it
very unlikely that the sweat gland extract potentiated
.
.
: , - . i . ,,,, ~ . ,. .. .. .. , . ;
WO92/11026 PCT/US91/09650
- 30 -
2~9~922
the cholinergic-inducing effects of the rat serum
present in normal growth medium (Wolinsky et al., 1985,
J. Neurosci. 5, 1497-1508; Wolinsky and Patterson,
1983, J. Neurosci. 3, 1495-1500).
Sweat gland extracts were tested for their
ability to support the survival of cultured sympathetic
neurons. Table II shows that neurons plated in medium
lacking nerve growth factor (NGF) but containing sweat
gland extracts at a dose of 1 mg/ml did not survive
10 more than 3 days in culture. Furthermore, there was no
significant difference in neuron number in cultures
grown with or without sweat gland extract even at
extract doses as high as 1 mg/ml in the presence of 50
ng/ml of NGF. Since the levels of choline
acetyltransferase activity and acetylcholine synthesis
are initially very low in dissociated sympathetic
neuron cultures: (Johnson et al., 1976, Nature 262,
308-310; Johnson, 1980, J. Cell Biol. 84, 630-691;
Patterson and Chun, 1977, Dev. Biol. 60, 473-481) and
20 there was no significant change in cell number with a
50 to 100-fold induction of chsline acetyltransferase
activity, it is extremely unlikely that the induction
of choline acetyltransferase observed in the presence
of sweat gland extract is due to the selective survival
25 f preexistlng cholinergic neurons.
.
' 35 ,
', ' ' '
.
'. '
WO92/11026 PCT/US91/09650
- 2l~98922 :::
TABLE II ,
Effect of NGF and Sweat Gland Extracts on Survival of
- Svm~athetic Neurons
Cell Number _ _ _
-NGF -NGF+ Ext +NGF +NGF + ~
~ ... .
10 Day 2 3027 + 35 4734 + 164 5027 + 231 5324 +
186
Day 5 10 + 12 12 + 13 4624 + 112 4867 + ::;
128
... .
, .
5 Sympathetic neurons were cultured in 56 well plates in
Ll5-CO2 with NGF (50 ng/ml) for 2 days. On the second ;
day, the culture medium was replaced with medium -
containing no NGF ~-NGF), no NGF but with l mg~ml sweat
i gland extract
(-NGF ~ Ext), 50 ng/ml NGF (+ NGF), or 50 ng/ml NGF
plus l mg/ml sweat gland extract (~NGF + Ext). Cells
1 20 were counted after an additional 3 days in culture.
I Samples were run in triplicate. Data are expressed as
the number of cells surviving per well ~ SEM.
i
6.l.2. CHOLINE ACETYLTRANSFERASE
INDUCTION IS DOSE DEPENDENT
Serial dilutions of sweat gland extracts were
added to cultured sympathetic neurons, and the wells
were assayed for: choline acetyltransferase activity 7
days later. Induction was seen with doses as lOw as l0
~ ~g/ml and increased with the addition of increasing
1~ amounts of extract to the maximum dose tested (Figure
; ~ 2a). When extract concentrations much higher than 1
mg/ml were used, some toxicity was evident in the
cultures: neuron number was reduced, and cell bodies ~-
i
~.~ - -. .. . :, , . , - . - , . ..
WO92/11026 PCT/US91/09650
2098922 - 32 -
were smaller in size. Toxicity may be due to high
concentrations of the cholinergic-inducing activity in
the extract, since similar effects of high doses of
other cholinergic-inducing factors have been described
(Fukada, 1985, Proc. Natl. Acad. Sci. USA 82, 8795- -
8799; Saadat et al., 1989, J. Cell Biol. 108, 1007-
1~16). Alternatively, it may be due to other compounds
in the preparation.
The time course of induction was also
10 determined. Elevated choline acetyltransferase
activity was detected as early as the second day in
culture and continued to increase through day 14, the
last time point assayed (Figure 2b). This time course
of cholinergic induction in sympathetic neuron cultures
is similar to that reported for heart and muscle cell
conditioned medium factors, presumably CDF/LIF
~Patterson and Chun, 1977, Dev. Biol. 60, 473-481;
'Raynaud et al., 1987, Dev. Biol. 121, 548-558), and for
CNTF (Saadat et al., 1989, J. Cell Biol. 108, 1807-
20 1816). In contrast, increased choline
acetyltransferase activity is seen significantly sooner
following treatment of sympathetic neurons with MANS
(Adler et al., 1989, Proc. Natl. Acad. Sci. USA 86,
1080-1083) or treatment of spinal cord cultures with a
25 soluble factor i5elated from skelstal muscle ~McManaman
et al., 1988, J. Biol. Chem. 263, 5890-5897).
6.1.3. SWEAT GLAND EXTRACTS CAUSE A REDUCTION IN
THE EXPRESSION OF NORADRENERGIC PROPERTIES
Not only does choline acetyltransferase
activity appear during the normal development of the
sweat gland innervation, but there is also a
concomitant reduction in tyrosine hydroxylase ~
immunoreactivity and catecholamine histofluorescence ~ -
(Landis and Xeefe, 1983, Dev. Biol. 98, 349-372; Landis
~
WO92/11026 PCT/US91/09650
~ 33 ~ 2~98 922
'' .
et al, 1988, Dev. Biol. 126, 129-140). If the sweat
gland extracts contained a factor(s) that played a role
in altering neurotransmitter phenotype, one would
predict that it would decrease the expression of
noradrenergic properties in cultured sympathetic
neurons. To assay the effect of sweat gland extracts
on tyrosine hydroxylase levels, equal protein aliquots
of neurons grown with and without extract were
su~jected to SDS-PAGE ~sodium dodecyl sulfate-
polyacrylamide gel electrophoresis), blotted onto
nitrocellulose, and probed with a monoclonal antibody
to tyrosine hydroxylase (Rohrer et al., 1986, J.
Neurosci. 6, 2616-2624; the kind gift of Dr. A.
Acheson, University of Edmonton). A single band was
evident at 62 kd, the expected molecular mass
(Lamouroux et al., 1979, Proc. Natl. Acad. Sci. USA 79,
3881-3885). Visual inspection of the immunoblots
suggested that the amount of immunoreactivity was
significantly reduced in cultures grown with sweat
20 gland extracts (Figure 3). When the color intensity
was read with a laser densitometer, cultures grown with
100 ~g/ml sweat gland extract exhibited a 2.5-fold
reduction in the level of tyrosine hydroxylase-
detected. In contrast, levels of immunoreactivity for
25 a cell surface adhesion molecule, L1 (Rathjen and
Schachner, 1984, EMBO. J. 3, 1-10), revealed with a
polyclonal antiserum (the kind gift of Dr. U.
Rutishauser, Case Western Reserve University) were not
reduced when assayed in a similar manner.
To determine whether the change in the level
of tyrosine hydroxylase was associated with a
corresponding change in the level of catecholamines,
the catecholamine content was determined in cultures of
sympathetic neurons grown with and without sweat gland
35 extract. The total catecholamine content of wells ;
-
~ - .. : , , . , . . ~
WO92tl1026 PCT/US91/09650
209~922 34 _ ~
-
incubated with sweat gland extracts was reduced
compared with that of control cultures (Table III).
TABLE III
Sweat Gland Extracts cause a Reduction in the -~
-Detectable Levels of Catecholamines
Catecholamine -
(pmol ~er Well~ % Reduction
Control medium 14.15 + 2.33
Sweat gland extract
A (11) 10.65 + o.g 23.3
B (26) 7.95 + 0.13 46.5
c ~47) 6.0 + 0.6 60
Sympathetic neurons were grown with sweat gland
extracts (100 ~g/ml, 250 ~g/ml and 1 mg/ml). Seven
~; days after the addition of extracts, the cultures were
harvested and assayed for catecholamine content by
high-performance liquid chromatography. Samples were
, run in triplicate. Data are expressed as mean pmol of
catecholamines per dish + SEM. The figures in brackets
20 are the mean fold choline acetyltransferse induction
assayed in sister wells by the method of Fonnum (1969,
Biochem. J., 115, 465-472).
..... ..
An inverse correlation was observed between choline
25 acetyltransferase activity and catecholamine content;
as the induction of choline acetyltransferase
increased, the catecholamine content decreased. This
relationship has been observed previously in studies
, with heart and s~eletal~muscle cell conditioned medium -
. . .
(Patterson and Chun. 1977, Dev. Biol. 56, 263-280;
Raynaud et al., 1987, Dev. Biol. 121, 548-Ss8). ~-
t
;~
.' -~.
, 35 ~;.,:,'.'.. ,
`2 ~ : .
~. .
-
W O 92/11026 P ~ /US91/09650
5 - ~09~922 : ~ :
6.1.4. SWEAT GLAND EXTRACTS ALTER
NEUROPEPTIDE EXPRESSION : .
Changes in neuropeptide expression are
observed in the developing sweat gland innervation.
VIP immunoreactivity is initially absent but becomes
5 detectable by P10; the immunoreactivity increases in
extent and intensity with subsequent development.
Since the sympathetic innervation of foot pads
transplanted to the thorax acquires VIP
immunoreactivity, sweat glands are able to induce VIP
10 expression in addition to choline acetyltransferase
activity tSchotzinger and Landis, 1988, Nature 335,
637-639; unpublished data). We therefore assayed
cultures of sympathetic neurons treated with sweat
gland extract by radioimmunoassay to determine whether
5 extracts increased VIP levels. Sympathetic neurons
grown in control medium contain relatively little VIP
immunoreactivity. Sweat gland extracts significantly
increased VIP ~Figure 4a); a dose of 100 ~g/ml causes
an induction of 80 pg per well of VIP, a more than 4-
20 fold increase over the levels present in controlcultures. The induction of VIP expression increased
with increasing concentrations of sweat gland extracts
~Figure 4a). ^,
The effect of sweat gland extract on NPY
content was examined because previous studies have
shown that while many noradrenergic sympathetic neurons
contain NPY immunoreactivity, cholinergic sympathetic
neurons, including those that innervate sweat glands,
do not tLandis, et al., 1988, Dev. Biol. 126, 129-140;
Lindh et al., 1989, Cell Tissue Res. 296, 259-273).
The content of NPY-like immunoreactivity was high in
control cultures, as observed in previous studies
(Marek and Mains, 1989, J. Neurochem. S2, 1807-1816;
35 Nawa & Sah, 1990, Neuron 4, 279-287). Growth in the
presence of sweat gland extract led to a reduction in
-, - ,: . , . - ,. - . ~ , .: .. ,, . .. :
WO92/11026 PCTIUS91/09650
~098922; - 36 -
NPY content (Figure 4b). This reduction is in marked
contrast to the elevation of VIP content and indicated
that sweat gland extracts regulate the levels of the
two peptides differentially. The decreased expression
5 of NPY in sympathetic neurons grown with sweat gland ~-
extract is consistent with results of a previous study
of target effect on peptide expression ln vlvo;
following transplantation of the superior cervical
ganglion from newborn rats to the anterior chamber of
10 the eye, NPY-lR was absent when the ganglion was
cotransplanted with sweat glands, but present when the
ganglion was cotransplanted with the pineal gland
~Stevens and Landis, 1990, Dev. Biol. 737, 109-124).
lS 6.l.5. AGE DEPENDENCE OF CHOLINE ACETYL-
TRANSFERASE-INDUCING ACTIVITY IN
EXTRACTS OF SWEAT GLANDS
The change in neurotransmitter properties in
the developing sweat gland innervation occurs
postnatally and is esentially complete by P21. To
20 determine the e~rl~est age at which detectable
cholinergic-inducing activity was present in developing
glands, extracts were prepared from footpads of animals
ranging in age from 2 to 21 days and were assayed for
their ability to induce choline acetyltransferase
activity (Figure 5). Increased levels of choline
acetyltransferase were detected in cultures treated
with extracts from P5 glands, and choline
acetyltransferase-inducing activity was present at all
subsequent ages. Less than a 2-fold difference was
30 evident in the specific cholinergic-inducing activity
present in footpads of P5 and adult animals when the
inducing activity was expressed as the amount of
choline acetyltransferase activity detectea per mg of
extract protein. The amount of protein extracted from
20 footpads, however, varied almost 15-fold from the
.:
.
-
WO92~11026 PCT/US91/096S0 ~.
- 37 - . .
'~098922
: - .
youngest to the oldest animals. Thus, the absolute
amount of choline acetyltransferase-inducing activity
increased approximately 30-fold during development.
These results indicate that cholinergic differentiation
activity is present in developing glands when the
properties of the innervation change. In addition,
extracts from sweat glands of P9, Pl4 and P21 rats were
found to increase the expression of VIP and decrease
the levels of tyrosine hydroxylase, as well as increase
choline acetyltransferase (data not shown).
6.1.6. INITIAL CHARACTERIZATION OF THE
FACTOR(S) RESPONSIBLE FOR CHOLINE
ACETYLTRANSFERASE INDUCTION
Preliminary characterization of the choline
15 acetyltransferase-inducing activity is summarized in -.
Table IV. -
;
. .
~ 25
.
, . .
., .. ~ '.
s 35
.;
.
, , , . - : - .: : -
'": ' ~'. ' ~. ' ' ' ~ :
~: ~ ' ' "' '. ' .:: ' : ' ' :
;-'. . :- :,: ' ' :- :- ' . . . : '.. .' .:: '- . ' : .: ~
W~92/11026 PCT/US91/09650 ~'
æog~92~ ~ 38 -
TABLE IV
Physicochemical Characterization of
Cholineraic Differentiation Activity
% Activity_~ tained
Thermal stability
-20C/-70C storage 90
Freeze-thawing 50
Boiling (100C for 5 min) 0
~rotease treatment
10 Trypsin o ,
Trypsin + i~hibitor 27
Heparin-agarose chromatography , ,
Flow-through 55 ' '
eluate 8' '
Centricon retention ''
lO kd cutoff 95
15 50 ~d cutoff 50
DEAE chromatography
Flow-through 2 ' , ,-
0.25 M elute 50 ,'
Aliquots of sweat gland extracts (lO0 ~g/ml) were
20 incubated as descri,bed below or in Experimental " "' Procedures. To examine the effects o~ protease
treatment, aliquots were incubated for l hr wlth
trypsin ~l mg/ml) or with trypsin and trypsin inhibitor
~3 mg/ml). F~ Centricon separation, samples were spun
in a SS34 rotor until the retentate volume was 25 ml. '
The retentate was diluted to l ml and spun again.
25 After three such spins, the flow-through was collected
and concentrated. Choline acetyltransferase activity
was determined in cultures 7 days after the addition of ,
treated extracts. A value of lO0~ represents activity
evident in cultures exposed to untreated ,extract and 0~ -
represents activity in cultures grown without the '
addition of extract. , - ,
' ' ''~ .
: . .
The activity was heat and trypsin labile and retained -
by a Centricon filter with a lO kd cutoff, indicating
35 that the activity is a protein. The activity was only ' "
partially retained by a Centricon filter with a 30 kd -
,. :.
.
:-:
. ,. ~ .. : . : . . : . , . - - -
. . ., j ,, - , . , . . , . . ., ...... . . . . - .
WO92/11026 PCT/US91/09650
- 39 -
2098~22
cutoff, suggesting that a low molecular mass protein is
responsible for the induction of choline
acetyltransferase. The cholinergic-inducing activity
was relatively stable; little activity was lost with
storage at -20C and on repeated freeze-thawing. Since
none of the activity bound to a heparin-agarose column,
the sweat gland cholinergic factor does not appear to
be a heparin binding protein like the 50 kd soluble
cholinergic factor from brain (Kessler et al., 1986,
0 Proc. Natl. Acad. Sci. USA 83, 3528-3532). Almost all
choline acetyltransferase-inducing activity and 35% of
the protein were recovered in the 0.25 M eluate from a
DEAE column, indicating that the differentiation
activity is an acidic protein(s) and that this can be
15 used as an initial purification step. The 0.25 DEAE
eluate not only induced choline acetyltransferase
activity, but also increased levels of VIP and reduced
levels of tyrosine hydroxylase (data not shown). Thus,
the several effects of the sweat gland extract on
20 neurotransmitter properties of cultured sympathetic
neurons are not readily separated.
I 6.1.7. FAILURE TO IMMUNOP~ECIPlTATE BIOLOGICAL
! ACTIVITY WITH ANTIBODIES TO CDF/LIF
one candidate ~or the cholinergic-inducing
25 activity in sweat gland extracts is CDF/LIF, since it -~
' has many of the same effects on the neurotransmitter
~ properties of cultured sympathetic neurons. Antisera
;~ generated against a synthetic peptide whose sequence
corresponds to the N-terminal peptide sequence of
~ CDF/LIF can immunoprecipitate the cholinergic-inducing
j activity from a partially purified DEAE fraction from
heart cell conditioned medium (Yamamori et al., 1989,
,, Science 246, 1412-1416; Rao et al., l99O, Dev. ~iol.
35 139, 65-74). When the DEAE fraction of the sweat gland
extract was treated with affinity-purified antibodies
.
: . ,.
; ...... , . . . ~, ~ ,. ,.,. .,...... , . , ,, ,. , ~ ,
- .. .- . . - . - ~ .
:: :- : i - . , . . .. ~ .
.. -., ,: : -,
WO92/11026 PCT/US91/09650
2~9~922 - 40 -
to CDF/LIF, there was no detectable decrease in the
~ability of the extract to induce choline -
acetyltransferase activity (Figure 6a). Since the DEAE
fraction is relatively crude, it was possible that
inhibitory proteins or proteases, present in the sweat
gland extract, were responsible for the inability to
immunoprecipitate activity. In parallel experiments,
however, the same antibodies added to the DEAE fraction
of sweat gland extracts were able to precipitate
10 iodinated recombinant CDF/LIF tthe kind gift of Dr. T.
Yamamori, California Institute of Technology) as the
appropriate 20 kd band (Figure 6b). Thus, the
principal cholinergic-inducing activity present in the
sweat gland extracts is not likely to be CDF/LIF.
6.l.8. NCDF IS DISTINCT FROM CNTF
Another candidate for the cholinergic
inducing activity in sweat glands was ciliary
neurotrophic factor ~CNTF). To examine this
20 possibility, equal amounts of cholinergic inducing
activity Srom sciatic nerve extracts and sweat gland
extracts were loaded on an SDS-PAGE gel,
electrophoresed and probed with a polyclonal antiserum
generated agilnst recombinant rat CNTF ~a k~nd gift of
25 Dr. Mark Furth, Regeneron Pharmaceuticals). The
antiserum recognized recombinant CNTF (Fig. 7a) and a
24 kd band present in the sciat1c nerve extracts (Fig.
7b). Binding was completely blocked by preincubating
the antibody with lO~m recombinant CNTF. No specific
30 band was evident in the lanes containing either hairy
skin extracts or sweat gland extracts even though the
` lanes containing the sweat gland and sciatic nerve
¦ extracts contained the same amount of cholinergic
i inducing activity for cultured sympathetic neurons (see
35 also Fig. lb). Loading ten-fold more cholinergic -
'
... .
. .
- . . - . .- .. - .. - . . . . . , . . . -
., ... ~ i "- .. : - . - ... : . ; - .. -.- :,,,
WO92/11026 PCT/US91/09650
- 41 -
'2098922
inducing activity and overstaining the blots failed to
reveal any specific binding in lanes containing sweat
gland extracts. Thus, the cholinergic inducing
activity present in sweat gland extracts does not
appear to be identical to CNTF.
.:
6.l.9. NORTHERNS AND IN SITU HY~RIDIZATION WITH
AN OLIGONUCLEOTIDE PROBE AGAINST RAT CNTF
To determine if message for CNTF could be
detected in sweat glands and to identify the cells that
10 may be prodùcing the CNTF/CNTF-like molecule, we
prepared RNA from the adult sweat gland and probed the
Northern blots for message with a probe against rat
CNTF (Fig. 8). A band of l.3 kb, the expected size of
CN$F message, was detected in sciatic nerves. In
5 contrast, no specific binding was detected in lanes
containing RNA from liver or sweat glands.
The same probe was also used to probe
sections of sc~atic nerve and sweat gland by in situ
hybridization, as a more sensitive assay of the cells
20 type that may be ma~ing CNTFjCNTF-like molecule.
Schwann cells in the sciatic nerve showed a specific
hybridization signal with the antisense probe as
compared to thé sense control ~Flg. 9). No speciric
signal however, could be detected in sweat gland tissue
25 ~Fig. lO).
6.l.l0. ANIONIC EXCHANGE CHROMATOGRAPHY
The 0.25 N DEAE eluate contained-the ChAT and
VIP inducing, and tyrosine hydroxylase reducing,
activity. Almost all activity and 35% of the protein
was recovered in the 0.25 M eluate of a DEAE column,
sugqesting that this can be used as an initial
purification step (Fig. ll).
:' ' " '. ` ',~ ' ' . ' . . ' . , ', ' , '' , ' .. , ' ,' '. . . . ~
W O 92/11026 P ~ /US91/09650 ~
~9~922 - 42 - ~.
6.1.11. ISOELECTRIC FOCUSING
The D~AE eluate was chromatographed on a MONO
P column and 0.5 ml fractions were collected and
assayed for cholinergic activity. Fig. 12 shows that
the ChAT activity was eluted at between pH 4.8 to 5.2
with a peak of activity at pH 5.0, indicating that the
pI of the active protein was in this range. This is
similar to the value reported for CNTF purified from
sciatic nerve extracts and differs from LIF which is a
10 strongly basic protein.
6.1.12. SIZE FRACTIONATION
To determine the molecular weight of the
cholinergic inducing activity, partially purified --
15 fractions were chromatographed on a sizing column. -
Fractions were tested for activity on sympathetic
neuron cultures. Almost all the activity eluted in a
, .
peak between 16 kd and 32 kd protein markers,
indicating that NCDF i8 a low molecular weight protein.
To more accurately determin~ the molecular
, weight of NDCF, fractions of the sweat gland extract
purified on a chromatofocussing column (Fig. 12) were
j run on a SDS PAGE gel, and bands of the appropriate
molecular weight were cut out a~d the proteins eluted
2S using an electroeluter. Aliquots of the extracted
proteins were tested for activity (Fig. 13). The
activity had a molecular weight between 22-26 kd (Fig. - -
13).
` The same fraction which had cholinergic
- 30 inducing activity was also tested for its ability to
modulate levels of tyrosine hydroxylase. The same SDS
PAGE eluted fraction had tyrosine hydroxylase reducing
~` activity.
s -:-
.':
..
WO~2/11026 PCT/US9t/09650
- 43 -
2~
6.2. DISCUSSION
We prepared low salt extracts of sweat gland
tissue and tested the ability of these extracts to
modify the neurotransmitter phenotype of cultured
sympathetic neurons. Extracts of sweat glands but not
of liver, hairy skin, or parotid gland increase levels
of cnoline acetyltransferase activity and of VIP-like
immunoreactivity in a dose-dependent fashion. As the
levels of choline acetyltransferase activity increase
10 in the cultured neurons, there is a concomitant
decrease in the catecholamine content and tyrosine
hydroxylase. ~hus, extracts of soluble protein from
sweat glands cause many of the changes in cultured
sympathetic neurons that characterize the developing
sweat gland innervation in v vo and that are induced by
the glands in cross-innervation experiments.
The ability to alter neurotransmitter
properties is present in sweat gland extracts of
animals between P5 and P21, when the fibers innervating
20 the sweat glands are changing from noradrenergic to
cholinergic (Landis and Keefe, 1983, Dev. Biol. 98,
349-372; Leblanc and Landis, 1986, J. Neurosci. 6, 260-
265; Landis et al., 1988, Dev. Biol. 126, 129-140).
Extracts from ~5 anlmals increase choline
25 acetyltransferase activity, and when extracts of glands
from animals between P9 and P21 are tested, they alter
all three transmitter properties examined: they
increase choline aceyltransferase and VIP-like
immunoreactivity and reduce tyrosine hydroxylase.
30 Furthermore, since elevated levels of choline
acetyltransferase activity are detectable after 2 days
of treatment in culture, the extract is able to induce
changes with a time course consistent with in vivo
studies. Establishing a more precise temporal
35 correlation is difficult, since the changes observed in
' :"
'' ' ' ~ "., ''. '''' ~ , ~ ' ' '' '" '. , .
WO92/11026 PCT/US91/09650 ~
2098~22 - 44 ~
the neurotransmitter properties of the terminal plexus
in the sweat glands in situ presumably reflect
retrograde transport of a target-derived signal,
altered expression of transmitter synthetic enzymes and
peptides, and anterograde transport of these molecules
to the terminals. --
Sweat glands of adult as well as developing
animals contain NCDF activity.
Although comparison of the levels of
b~ological activity observed ~n vitro with those
present 1n Yivo is difficult, it is of interest to
estimate whether the glands contain enough cholinergic-
inducing activity to mediate the switch. Retrograde
tracing studies suggest that at least 200 neurons
innervate the six palmar pads (Siegel and Landis,
unpublished data). our extraction procedure yields
about 10 mg of soluble protein per gram of footpad -
~ tissue from 21-day-old animals, or approximately 80 ~g
¦ per single pad. Since choline acetyltransferase
20 activity is induced at concentrations as low as lQ
~g/ml in cultures containing several thousand neurons, `
it appears that they do contain sufficient quantities
o~ cholinerglc-inducing activity. The concentration of
- chollnergic-inducing activity present ln sweat gland
25 extracts is greater than that in spinal cord extracts
(Wong and Kessler, 1987, Proc. Natl. Acad. Sci. USA 84,
8726-8729; Adler et al. 1989, Proc. Natl. Acad. Sci.
USA 86, 1059-1083) and at least as high as that in
sciatic nerve extracts (Sendtner et al., 1989, Soc.
Neurosci. Abs. 15, 710; Rao et al., 1990, Dev. Biol. -
139, 65-74).
Since CDF/LIF (Fukada, 1985, Proc. Natl.
Acad. Sci. USA 82, 8795-8799; Yamamori et al., 1989,
- Science 246, 1412-1416; Nawa and Patterson, 1990, :
Neuron 4, 269-277) and CNTF (Sendtner et al., 1989,
.' ' . ~, ;,. .
: .
.
WO92/11026 PCT/US91/09650
2098~22
Soc. Neurosci. Abs. 15, 170; Ernsberger et al., 1989,
Neuron 2, 1275-1284) cause the induction of cholingeric
function, the reduction of catecholaminergic function,
and an increase in VIP expression in sympathetic neuron
cultures, it is clear that a single molecule can affect
changes in all these properties. Two observations from
the present studies are consistent with the notion that
one molecule in the extracts is responsible. Extracts
prepared from animals of different ages influence the
1O several properties assayed, and more importantly, the
several effects were not resolved into distinct
; activities in the preliminary characterization that we
; have performed. Thus, a single molecule seems likely;
however, the possibility that several factors are
15 involved cannot be formally excluded.
It is of interest to compare the properties
~ of the activity in sweat gland extract with the several
.J ~ factors that have been prev~ously described to induce
cholinergic function in cultured sympathetic neurons.
20 Since the cholinergic-inducing activity present in the
sweat gland extracts is easily extracted in low salt
; solutions and no detectable activity is associated with
membranes (unp~blished data), it is not likely to be
~ relatsd to the membrane-associated factors that induce
25 choline acetyltransfera-~e ~Wong and Kessler, 1987,
Proc. Natl. Acad. Sci. USA 84, 8726-8729; Adler et al.,
1989, Proc. Natl. Acad. Sci. USA 86, 1059-1083) and
I reduce levels of tyrosine hydroxylase (Rap et al.,
1990, Dev. Biol. 139, 65-74; Lee et al., 1990, Exp.
30 Neur. 108, 109-113) in cultured sympathetic neurons.
~ In addition, there is a difference in the time course
J of induction of choline acetyltransferase activity;
-~ cultures treated with sweat gland extracts exhibit a
small increase of activity after 2 days, whereas
35 cultures treated with a membrane-associated -
-~
. , .
.. . ~.
- ' .
:` : : . -. ' . . '::, ', . . ~ . ' :' : ` . ' ' . : ..
WO 92tl1026 PCT/US91/09650
8922 -46- ~
cholinergic-inducing activity show high levels of
activity in the same time period (Adler et al., 1989,
Proc. Natl. Acad. Sci. U.S.A. 86, 1080-1083). Two
soluble factors, CDF/LIF and CNTF, are similar in their
5 effects on sympathetic neurons; they increase choline
acetyltransferase and VIP expression and reduce
tyrosine hydroxylase and catecholamine content (FuXada, -
1985, Proc. Natl. Acad. Sci. USA 82, 8795-8799;
Yamamori et al., 1989, Science 246, 1412-1416; Sendtner
10 et al., 1989, Soc. Neurosci. Abs. 15, 710; Ernsberger
et al., 1989, Neuron 2, 1275-1284; Nawa and Patterson,
1990, Neuron 4, 269-277). In addition, like sweat
gland extract, both CDF/LIF (Nawa and Patterson, 1990,
Neuron 4, 269-277) and extracts of sciatic nerve `
containing CNTF (unpublished data) decrease NPY
expression. ~ -
CDF/LIF was an attractive candidate; it has a
consensus signal sequence, it is glycosylated, and it
is secretad by heart cells (Patterson and Chun, 1977,
20 Dev. Biol 56, 263-280; Yamamori et al., 1989, Science
246, 1412-1416). CDF/LIF, however, does not bind to a
DEAE column ~Fukada, 1985, Proc. Natl. Acad. Sci. USA
82, 8795-8799), unlike the cholinergic-inducing
activity in s~eat glands. Furthermore, affinity-
25 purified antibodies raised against the N-terminal
region of CDF/LIF can immunoprecipitate the
cholinergic-inducing activity from the DEAE or Sephadex -
fractions of heart cell conditioned medium (Yamamori et
al., 1989, Science 246, 1412-1416; Rao et al., 1990,
30 Dev. Biol. 139, 65-74), but these antibodies do not
immunoprecipitate the cholinergic-inducing activ~ty
from sweat gland extracts. Thus, it is unlikely that
the major cholinergic factor in the extract is
identical to CDFlLIF. ~ 1
; -
I
:... .
: .: . . .~ . . . . . , . .. . - ' . . . ~ . .
.
WO92/11026 PCT/US91/09650 ~ -
209~3g22
CNTF was another likely candidate. It is an
acidic protein and can be eluted from a DEAE column by
0.25 M NaCl ~Manthorpe et al., 1980, Neurochem. 34, 69-
75; Manthorpe et al., 1986, Brain Res. 367, 282-286;
5 Barbin et al., 1984, J. Neurochem 43, 1468-1478), much
like the cholinergic-inducing activity present in the
sweat gland extracts. However, Northern blot analysis
of adult glabrous skin containing sweat glands failed
to demonstrate detectable message for CNTF, whereas
10 abundant message was present in sciatic nerves ~Stockli
et al., 1989, Nature 342, 920-923; Sendtner et al.,
1989, Soc. Neurosci. Abs. 15, 170) even though the two
tissues contain similar levels of cholinergic-inducing
activity. In addition, immunoblot experiments with a
polyclonal antiserum generated against and recognizing
recombinant CNTF failed to reveal any CNTF-like
s immunoreactivity in sweat gland extracts. Furthermore,
;~ Northern blot and in situ hybridization assays failed
to reveal any specific hybridization ln samples from
20 sweat glands with a rat CNTF probe. These data,
~ together with the fact that CNTF appears to be a
f cytosolic protein (Stockli et al., 1989, Nature 342,
j 920-923; Lin et al., 1989, Science 246, 1023-1025);
while a candid~te sweat gland differentiation molecule
;` 25 ~s likely to be secreted to exert an effect on the
innervation, suggest that CNTF is an unlikely candidate
~i for the sweat gland-derived cholinergic factor.
In summary, we have shown that a cholinergic
. sympathetic target tissue, sweat glands, contains
30 cholinergic differentiating activity that mimics in
culture the effects of the target on sympathetic ~ -
s neurons in vivo. Our preliminary purification and
analysis suggest that the cholinergic-inducing activity
- present in the sweat gland extracts is not identical to
35 either CDFILIF or CNTF and that it is a no~el factor. --
. ` . . .
.~ .
' . : .
:. ~ . , ., . , . ., . . . ~ ~ . -
:. - . - - : . . . . ... . . . . ..
W O 92/11026 ps~T/US91/09650
~og~922 - 48 ~
Thus, the cholinergic-inducing activity present in
sweat gland extracts represents an excellent candidate
for mediating the target-induced phenotypic changes in
the cholinergic sympathetic neurons that innervate
5 sweat glands.
, ,.,~, . ..
6 . 3 . EXPERIMENTAL PROCEDURES~ -
6.3.l. MATERIALS
Cell culture reagents were obtained from
GIBCO (Grand Island, NY) and culture plates were from
Corning ~Corning, NY). The Centricon filters were
purchased from Amicon (Danvers, MA). ~3H~-acetyl-CoA -
and Bolton Hunter reagent were purchased from New
England Nuclear (Wilmington, DE). Dispase was obtained
15 from Boehringer Mannheim (Indianapolis, IN), and
collagenase was from Worthington Biochemicals ;
(Freehold, NJ). VIP radioimmunoassay kits were
obtained from Incstar (Stillwater, MN), and NPY
radioimmunoassay reagents were from Amersham. NGF (the
' 20 kind gift of Dr. K. Neat, Case Western Reserve
Univer~ity) was prepared from male mouse submaxillary
glands as described by Bocchini and Angeletti (1969,
Proc. Natl. Acad. Sci. VSA 64, 787-794). Pierce
protein assay ~it was obtained from Pierce (Rockford,
25 IL). ITS Promix was ~rom Collaborativ~ Research
tBedford, MA) and reagents for SDA-PAGE were from ~io-
' Rad (Richmond, CA). Avidin-conjugated alkaline
! phosphatase was obtained from Cappel (Westchester, PA),
and goat anti-mouse and anti-rabbit secon~ary
30 antibodies were from Jackson Immunologicals tWestgrove,
PA). Other chemicals were purchased from Sigma (St.
Louis, MO).
6.3.2. CELL CULTURE
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W O 92/11026 PCT/US91/09650
- 49 - ~209~922
Cultures of rat sympathetic neurons were
prepared as described by Hawrot and Patterson (1979,
Meth. Enzymthol. 58, 574-583). Neurons from the
superior cervical ganglia of newborn rats were
5 dissociated enzymatically with Dispase (5 mg/ml) and
collagenase (1 mg/ml and plated in 96-well plates
coated sequentially with polylysine (Q.l mg/ml) and
laminin (10 ~g/15ml) About 2000-3000 neurons were
plated per well except where indicated. The neurons
10 were grown in Leibovitz's L15-C02 medium with NGF (100
ng/ml), 100 U of penicillin, 100 ~g of streptomycin, 10
~M cytosine arabinoside, and 5~ rat serum, and the
medium changed every third or fourth day. In some
experiments, cells were grown without rat serum in L15- --
C02 supplemented with transferrin, selenium, bovine
serum albumin, insulin and fatty acids.
The tissue extracts were diluted in growth
medium, sterilized ~y passage through a 0.2 ~m filter
and added to the neurons from the third day of culture
20 on. Neurons were harvested for assay between days 9
and 14 of culture.
,:
6.3.3. TISSUE EXTRACTS
To p~epare 6weat gland extracts, footpads
25 were extracted ~rom rat~ of various postnatal ages and
weig~ed. Tissue from 20 animals was generally
processed at one time. The weight of footpads from 20
rats varied from 0.5 to 5 grams, depending upon the age -
of the animal. The tissue was homogenized for 5 sec in
30 10 vol of 10 mM phosphate butter (pH 7.0) with a
Polytron. The extract was then centrifuged at loo, ooo -
x g for 1 hr. The supernatant was collected, filtered
through a 0.2 ~m filter, and concentrated using a
Centricon filter with a-10 kd cutoff. The protein
35 concentration was determined with a Pierce protein
' : ,'':
.
WO92/11026 PCT/US91/09650
- 50 - -
2~9~922
assay kit. Extracts of liver, sciatic nerves and
parotid glands were prepared in a similar manner. To
prepare hairy skin extracts, the skin over the thoracic
region was shaved and dissected free from the
underlying panniculosis carnosus muscle and wei~hed.
The skin was then cut into smaller pieces before being
homogenized and processed as described above.
.
6.3.4. ASSAYS
The induction of cholinergic function was
determined by assaying choline acetyltransferase
activity in homogenates essentially according to the
method of Fonnum (1969, Biochem. J. 115, 465-472). To
increase the sensitivity of the assay, an incubation ;
1speriod of l hr was used. All activity was inhibitable -
'by 500 ~M napthylvinyl pyridine, a specific inhibitor
of choline acetyltransferase activity. Protein
concentration was assayed by the method of Lowry using
bovine serum albumin as a standard.
Catecholamine content was assayed by high
performance liquid chromatography ~Rittenhouse et al.,
1988, Neurosci. 25, 207-215) on a 5 ~m pore reverse-
pha0e C-18 column (Altex Ultrasphere-IP; Beckman,
Berkeley, CA) ~sing a colorometric detector (5100A,
; 25 ESA, Bedford, MA~. Three electrodes were set in series
at +0.36, +0.03, and -0.38 V relative to a reference
electrode. Standards at known dilutions (5 pmol~ were ~ -
run at the same time to estimate the concentration.
The total catecholamine content of a well was obtained
30 by summin~ the levels of norepinephrine, dopamine, and
DOPAC, a metabolite present in each extract. Neither
epinephrine nor DOPA was detected.
The amount of tyrosine hydroxylase present in ~
the cultured neurons was determined,by semiquantitative --
35 analysis of immunoblots. Cell cultures were
.,
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WO92/11026 PCT/US~1/09650
- 51 -
209~22
homogenized in sample buffer (50 mM Tris (pH 6.8) with
2% SDS, 10% glycerol, 0.004% bromophenol blue, and 3%
~-mercaptoethanol), aliquots of the extract were run on
a 10% SDS-PAGE gel, and the proteins were blotted onto
5 nitrocellulose. The nitrocellulose blots were blocked
in blocking buffer (5% defatted mil~ in Tris-buffered
saline [pH 7.2~) and then incubated with a monoclonal ~-
antibody against tyrosine hydroxylase (the kind gift of
Dr. Ann Acheson, University of Alberta, Edmonton)
10 overnight. The blots were then sequentially incubated
with a biotinylated secondary antibody and avidin
conjugated to alkaline phosphatase. The reaction
product was developed with Nitro Blue Tetrazolium and
5-bromo-4-chloro-3-indoyl phosphate in lO mM
15 bicarbonate butter (pH 9.5). After optimal color
development, the reaction was stopped by rinsing in
distilled water. The blots were allowed to dry, and
the color intensity was read on a scanning laser
densitometer (Shimadzu). -Comparisons were made between
20 samples run in parallel lanes and treated identically.
Neuropeptide levels were determlned by
radioimmunoassay. Cultures were rinsed once with PBS
and then homogenized in lO0 ~l of 2 M acetic acid.
After boiling ~or 5 min, samples were centrifuged for l
25 min in an Eppendorf microfuge. The supernatants were
dried under vacuum and stored at -70C for subsequent
assays. VIP was assayed using a kit obtained from
INCSTAR with primary antibodies previously demonstrated
to show minimal cross-reactlvity with other peptides.
30 To assay NPY by radioimmunoassay, antibodies, -
standards, and labeled tracer were obtained from -;
Amersham and peptide content was determined by the
delayed tracer method. Since the antibody shows only
64% cross-reactivity with rat NPY, standards were also
.
~ `. -: . - . : ' : . . , :: ~ -. . .
WO92/11026 PCT/US91/096~50
2~98922 - 52 - -
run with rat NPY (Peninsula Laboratories) and sample
values were read from the standard curve.
6.3.5. DEAE CHROMATOGRAPHY
Aliquots of the soluble extract were diluted
5-fold with l0 m~ phosphate buffer (pH 7.0~ and applied :
to 0.9 X l0 cm DEAE column (Whatman/Bioprobe) at a flow
rate of l0 ml/hr. The column was washed with an equal
volume of phosphate buffer. The wash and flow-through
0 were pooled and concentrated using a Centricon filter
with a l0 kd cutoff. The bound protein was eluted with
l0 ml of 0.25 M NaCl and concentrated in a similar
manner.
6.3.6. HEPARIN A CHROMATOGRAPHY
Aliquots of the soluble protein were diluted
with buffer (lO mM phosphate, 150 mM NaCl (pH 7.2) and
applied to a heparin-agarose column. Bound protein was
eluted with 5 M NaCl. The efficacy of column bindinq
20 was tested using ~25I-labeled basic fibroblast growth
factor (a kind gift of Dr. J. Unnerstall, Case Western
Reserve University).
~ . .
6.3.~. IMMUNOPRECIPITATION EXPERIMENTS
WITH CDF/LIF ANTIBODIES ``
For immunoprecipitation experiments in which
the biological activity of the factors was tested,
aliquots of tissue extract sufficient for a cell - -
culture assay were added to buffer (PBS [pH 7.3] with
' 30 2% bovine serum albumin, 0.2% Triton X-l00, and 0.02~
PEG 6000). Affinity-purified antibodies raised!against
a synthetic peptide corresponding to the N-terminal
region of CDF (Rao et al., l990, Dev. Biol. 139, 65-74)
were added to each vial to a final concentration of l0
35 ~M. After an overnight incubation, the antigen-
antibody complex was adsorbed to l0 ~l of protein A-
W092/11026 ~ U 9 8 9 2 2 PCT/US91tO9650
Sepharose for an additional 2 hr at room temperature.The bound complexes were separated by centrifugation,
and the supernatant was diluted into Ll5-CO2 medium and
used for cell culture assays. Two controls were -~
5 performed to ensure that the loss of activity
consequent to absorption was due to a specific effect
of the antibody. Aliquots of extract were incubated
without the antibody and treated as described above,
and other aliquots were treated with antibody that had
10 been previously absorbed with 50 ~M synthetic peptide
originally used as antigen.
ln other experiments, CDF/LIF (a kind gift of
Dr. Yamamori, California Institute of Technology) was
iodinated with Bolton Hunter reaqent as described
15 previously (Rao et al., l990, Dev. Biol. 139, 6S-74).
About 20,000 cpm were added to buffer or an equal
volume of the DEAE fraction of the sweat gland extract
and immunoprecipitated with the N-terminal antibody as
described above. The counts that were
t 20 immunoprecipitated were extracted and analyzed by SDS-
PAGE.
~ , , .
6.3.8. WESTERN ~LOT EXPERIMENTS WI~H CNTF ANTISERUM
Aliquots of extracts ~60 ~g/lane) were ru~ on
25 a 15% SDS PAGE minigel (Biorad) and the proteins were
` blotted onto nitrocellulose. The nitrocallulose blots
were blocked in blocking buffer (S% defatted milk in
Tris buffered saline, pH 7.2) and then incubated for
two hours with a polyclonal antiserum raised against
' 30 recombinant rat CNTF (l:lOOO dilution; the kind!gift of
`~ Dr. Donna Morrissey, Regeneron Pharmaceuticals) or with
the antibody preincubated with lO ~M CNTF (Regeneron
Pharmaceuticals). The blots were then sequentially -
~ incubated with a biotinylated secondary antibody for an - -
j 35 hour and then with avidin-conjugated alkaline
,` ' .
, ,.
WO92/11026 2 0 9 8 9 2 2 PCT/US91/096~0
- 54 -
phosphatase for 30 min. The bound enzyme was detected
with Nitro Blue Tetrazolium and 5-bromo-4-chloro-3
indoyl phosphate in 10 mM bicarbonate buffer, pH 9.5.
After optimal color development, the reaction was
5 stopped by rinsing in distilled water.
. ':
6.3.9. NORTHERN BLOTS AND IN SITU HYBRIDIZATION
For Northern blot hybridzation, total RNA was
prepared from liver, sweat gland and sciatic nerve
using the single step guanidinium-isothiocyanate method
(Chomczynski and Sacchi, 1987, Analytical Biochem. 162,
156-159). 30 ~g of total RNA was loaded per lane and
transferred to a Genescreen nylon membrane. Blots were
probed with a 45 base pair oligonucleotide probe
15 (region 99-144) against rat CNTF radiolabelled with 32p,
Blots were sequentially washed and then examined by
autoradiography.
The same probe used in the Northern blot
experiments (except that it was labelled with
20 3~S~dA~P) was used for the in-situ hybridization
experiments as described (Siegel, 1989, Methods in
Neùroscience, ed. Conn P.M., Academic Press 1, 136-
150). In brief, fresh frozen sections of tissue were
fixed in 4% ~ormaldehyde ~or t~n minutes, and rinsed
25 twice in PBS slides, air dried and processed for
hybridization. Hybridization was performed at 42C for
fifteen hours in a humidified container using 107 cpm/ml
of probe. After washing the sections were dipped in
Xodak NTB-3 emulsion and exposed for 6 weeks. Sections
30 were developed, fixed and counterstained with ethidium
bromide.
Various modifications of the invention in
addition to those shown and described herein will
35 become apparent to those skilled in the art from the
'
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WO92/11026 PCT/US91/09650 - -
~ 55 ~ 20~8922
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foregoing description and accompanying drawings. Such
modifications are intended to fall within the scope of
the appended claims.
Various references are cited herein, the ~ :
6 disclosures of which are incorporated by reference
herein in their entireties.
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