my role today is to describe the XML transport
systems or so called ectoplasmic flow
and the role of these systems in the normal maintenance of motor
neurons.
And then to review what's known about external
transport in motor neuron pathology.
Now on the first side mm this is
a highly schematic representation of
a motor neuron
and in general it can be divided into three
parts a cell body,
an axon and the synaptic
terminals.
Now,
you all know that the cell body and the synaptic
terminals are the business ends of the
motor neurone.
The cell body with its dendrites,
which are not shown here are the area where
uh the interplay
with uh segmental and super segmental
influences occurs.
The motor nerve terminal is the
site of transmission too.
Uh the effect er in this case muscle
at the neuromuscular junction.
What's not showing on this diagram is a really proportion
of sizes of these structures,
the axon intervening between the cell body and the motor
terminal is actually
uh in a human motor neurone
several 100 times the volume of either
the cell body or of course the terminals.
And yet one of the striking facts in neurobiology is that the
axum is almost entirely incapable
of synthesis of such essential substances as
proteins.
Rather,
the Exxon depends on
uh the continuous delivery to
it of uh proteins
synthesized in the nerve cell body and
carried by external transport.
The cell body is consequently one of the most vigorous sites of
protein synthesis in the body.
In fact,
we probably should regard a normal cell body as a
protein.
Secretary sell engaged in the internal secretion of
protein and lack of proteins into the
axon and the means by which these substances are
moved within the axon are the accidental transport system.
So,
I'm talking about transport.
We're talking about the cell
body synthesis of external substances
in their movement within the axon.
At the present time,
we recognize three external transport systems which are
summarized on the slide.
Two are engaged in moving material away from the cell
body.
Fast anterograde and slow anterograde
transport,
and one retrograde transport moves
material toward the cell body.
Well briefly describe these
systems of external transport and then return to our
diagram attic uh neuron and fill
in some of the pieces.
Fast anterograde transport moves materials down the
Accident with a rate of about 400 millimeters a day.
It's worth reflecting that in the human psychotic nerve.
That means it will take roughly three days for newly synthesized
protein to reach the motor nerve,
ending this rapidly transported material
is associated in transport with sacks
of smooth and a plasmid particular.
Um As we discuss in a moment,
transmission related enzymes are among the
rapidly transported constituents that is cold master
race,
dopamine,
beta hydroxy Alice and other
enzymes involved in synaptic transmission.
Yeah,
these rapidly transported materials are preferentially
distributed to the synaptic terminal and
probably served to renew the member list components of the
terminals,
the synaptic vesicles and the axle M.
O.
And as we'll see,
the mechanism of fast transport is unknown,
but micro tubules appear to be involved.
Slow transport on the other hand,
Moves materials at a discreet rate of 1 - two
a day.
With still transport.
The materials carried are much better characterized
and consists primarily of the bulk of
ectoplasm that is micro tubules and neural
filaments uh Instead of being
preferentially distributed to the terminals,
solar transported materials are quite generally distributed
along the axon and their role is
a presumably the renewal of
these.
X surprise my constituents
finally,
retrograde transport carries materials toward the
cell body.
Uh And as we'll see,
these are both recycled materials delivered
to the ending by fast transport and exogenous
substances taken up in the synaptic cleft.
Uh We'll discuss in a moment recent
results suggest that the rate of retrograde transport should be
regarded as 250 millimeters per
day.
Now,
the next slide,
the next slide will show these systems in the
context of our schematic nirvana.
Yeah.
First,
with regard to fast transport
i in studies using
protein or glycoprotein precursors
injected near the cell body.
E.
M.
On choreographic and biochemical studies have
shown the presumptive
uh victoria will
uh synthesis and transport of these
materials uh
loosen injected near a motor neuron cell body for
example,
is incorporated into proteins which
will be rapidly transported
uh in rough and a plasma
particular um A
complement of these Are conjugated.
two sugars in the golgi apparatus
informed lack of proteins.
Both the non glock
oscillated proteins and the glycoprotein.
Then inter fast transport for the most part as
member list components of smooth and the plasma,
particularly other
substances such as cold as to raise our probably
within the smooth.
And applies in particular these
sacks or long to bills of ectoplasmic
particular.
Um then enter the transport
process and it's we look at the next
side,
we can see that a
if we take this as a rat
lumbar,
eventual horn and imagine
that we have just injected treated,
losing through micro pipettes into this region
15 minutes later,
essentially no radio activity can be found out
along the nerve.
Uh the position of radio activity
along the nerve indicated on the X axis by
three hours.
However,
a discreet front of radio activity has moved
out,
Reaching about 50 in
terms of 24 hours,
that would be about 400 a day
By six hours.
This materials reached about 100.
So,
at the rate of fast transport,
as we have indicated,
measured by The fastest moving front
of newly synthesized protein is about
400 a day,
there's a good deal of confirmatory evidence indicating that endogenous
enzymes a similarly move at that rate.
Uh This rate probably applies not
just to peripheral neurons but to the central nervous system as
well.
Uh Now,
if we go back to the previous
slide at the level of
the motor nerve ending,
it seems most probable that these
transported sacks and vesicles of smooth
enterprise in particular um provide precursors
for synaptic vesicles indicated
here and in addition are capable
of merging with the axon terminal
itself to add new Excel Emma to
the synaptic terminal
and elegant E.
M.
Auto radiographic studies by drones have
very firmly confirmed that idea
now.
Uh if we turn to slow transport,
uh we can first of all say that if the same sorts of
techniques are used,
protein precursor is injected near the cell body.
That uh the synthesis
of solar transported materials appears to take place
primarily on polly ribosomes.
And as we've indicated,
the primary substances synthesized are micro tube
bills and narrow filaments.
Uh whether these uh inter
transport as a solid uh continuous
fibers,
protein or a soluble subunits is not yet
defined.
I these materials,
as we said before,
are distributed generally along the axon.
So that is indicated on the next slide,
The amounts of radio activity one finds as one goes
distantly decrease.
It's also worth emphasizing that the rate
at 1-2 mm a day.
Presuming that that's applicable to the human means that
in a human psychotic nerve,
we would be talking about roughly three years before
uh a fresh bullets of newly
synthesized slowly transported protein would reach
uh motor nerve endings in the foot.
Oh and
now,
if we turn to a retrograde transport
again for a moment
uh we're here depicting
the into psychosis
of uh
membrane uh at the synaptic terminal
uh forming again,
sacks or tubules which are very similar to
the s er that has carried anterograde
which then enter a retrograde
transport.
Now,
uh the use of radio chemicals has
been more difficult in
establishing rates of transport and retrograde
direction because unlike
newly synthesized proteins in the cell body,
a very rapid entry of a
front of material doesn't occur with
retrograde transport.
If an IUD nated preformed protein is
injected into muscle up,
take it slow and one simply sees a
gradation of
transported activity along the nerve.
We've recently used a technique
for identifying the fastest
moving components of retrograde transport and have taken advantage
of the fact that cold,
among other things,
reversible e stops both fast and low
grade and retrograde transport.
So,
by putting a cold cuff on rats,
psychotic nerves and injecting an isolated protein in this
case I donated tetanus toxin into
the hind foot muscles.
Uh We have allowed a substantial accumulation of
radio activity to occur distal to the
cold block.
Then the cold block is rapidly re warmed
in the front of transported material
continues to move retrograde up the axon by doing
that dr Lloyd shield in our laboratory has
a established both in
vitro and in vivo,
A very predictable and constant transport rate of
about 200 and 50 millimeters a day.
Uh 242,
mm A day.
So that in many ways this
retrograde transport system can be looked on as the
obverse of anterograde transport.
Uh We imagine rapidly transported membrane
carried to the Exxon terminal inserted into Excel
Emma and at some later time
into psychosis producing
new member nous packages which are
returned by retrograde transport.
The one edition being that exogenous materials from the synaptic
cleft are capable of being carried back to the cell body.
Now,
later phenomenon probably has uh
considerable biologic and pathologic
significance.
We've indicated that tetanus toxin can be carried
retrograde and work with dr Donald
Price.
Uh Uh huh.
Uh We think it's most likely at the present time that the
route of entry of tetanus uh
into um motor neurons and the pre
synaptic region or tetanus acts
in clinical tetanus is via retrograde
transport.
Uh similarly uh nerve growth
factor and botulinum toxin appear to be carried
retrograde through the same sort of
system.
And finally it's very likely that a number of viruses and pathologic
significance including uh polio viruses
can ascend retrograde into the nervous system.
So that retrograde transport might be looked on if you will as
a back door to the central nervous system and
possibly quite important means of
entry of a
ideological path genetic factors in disease.
So that to summarize what
we've said,
we've talked about a a
rapidly transported.
Uh we talked about rapid transport
of members elements from cell body to terminal
from terminal to sell body.
And we think of fast anterograde and retrograde transport
as essentially providing a high speed shuttle system
between uh sell body in synaptic terminal.
On the other hand,
still transport provides a means of renewing
the enormous amount of X applies um between
so we have
briefly and obviously highly schematically summarized
these elaborate systems which neurons have evolved for maintenance of their
axons.
We want now to focus on what's known about
the role of these transport systems
in a path of
biology or pathology of motor neurons.
We should begin by emphasizing that at the moment only
one process in only one
process is there a clear cut relationship between
accidental pathology,
excellent generation and external transport.
And that situation is in acute watery and degeneration of
the axon while randy generation.
We mean the prompt degeneration,
the breakdown and ultimate figure psychosis of the axon
that occurs after.
For example,
cutting a nerve that occurs in the distance,
dump the excellent segments separated
from continuity with the cell body.
And a variety of
approaches have indicated
that the most important factor in initiating this
process of orange generation is the acute
complete interruption of accidental transport.
Inferential evidence suggests that it's fast
axonal transport.
This most important if one
calculates the time to failure to fire.
Of a no
muscular preparation with a
very distal external section
and then increasingly lengthens the
proximal,
increasingly lengthens the distal stump
and measures the time to failure to fire.
one finds that each additional 15 millimeters
of distance dumped length
provides physiologic
activity,
the capacity to transmit across the
neuromuscular junction for about one additional
hour.
Uh Similarly,
the length of the distal stump affects
the development of pathologic
changes of orange generation in the
Exxon.
Uh in that 15 mm/h correlates very
closely to the rate of fast
external transport.
If uh excellent transport is interrupted not by
excellent section,
but by application of culture seen or been
blasting along the course of the nerve or by a
sustained cold block.
A similar polarity generation of the
distance stump occurs.
Having said that we really have summarized all that is
known firmly about the relationship between
transport and excellent pathology.
I'd like to turn to one kind of chronic axillary
generation which has attracted our interest in the interest of a
number of laboratories
recently.
And that's the process of external dying back.
Now dying back today,
at least should be taken to indicate
a situation in which the axon
degenerates in a distant a proximal
fashion chronically.
That is if one
imagines the if one imagines
the axon making contact
with the neuromuscular junction.
Uh normally one can say in Warren degeneration
that the accident was sectioned in the distance trump rapidly
disappears in dying back.
One can document a very slow,
gradual process of
distal to proximal degeneration
so that I uhh
we're talking about a chronic degenerative process,
which is certainly applicable to a number of human peripheral
neuropathy.
Now,
the hypothesis that has
been raised regarding this
can be summarized as follows.
If we say that an acute interruption of external transport
produces acute degeneration of the distance dump of
the axon.
Uh then can't we imagine that a chronic
partial impairment uh in the
amount of transported material received by the
axon might lead to
a chronic excellent regeneration
decrease in transport could certainly occur either
because of fear interference
with cell body synthetic functions or
because of abnormalities and transport per
se.
And we would imagine that larger or
longer axons might be preferentially vulnerable
simply because they have more access to maintain that the endings of
their axons would represent,
if you will,
the last field of of irrigation.
Uh this has
led to a hypothesis
uh that really goes back at least 100 years
and was very clearly articulated by
1895.
Actually enough with regard to amyotrophic lateral sclerosis.
And the hypothesis is simply that nerve cell body
synthetic uh deficiencies
lead to just like some only
generation.
Mhm.
Well,
in our laboratory and as I indicated in other laboratories
recently,
we've begun testing this hypothesis using
experimental toxic neuropathy.
The disorder that we have
investigated most is
acrylamide intoxication in the rat.
We've also uh more recently been investigating
intoxication with 25 hexane dyan
in collaboration of dr peter spencer at Albert Einstein
acrylamide induces a
dying back neuropathy.
This animal was intoxicated for about eight weeks with a
chrome ride in his feed.
Mhm.
And uh
you can see that there is marked weakness
of defeat particularly
uh and uh a lesser
degree of weakness uh is president although it's not obvious
from the side.
Uh more approximately sensory
loss is uh
distant predominant as well so that these animals are not
only weak distantly but they have impaired sensation and
difficulty walking for that reason.
Now is this dyslexic degeneration the result
of diminished delivery of
transported constituents to nerve
terminals?
Well,
to investigate that question,
we've used a preparation,
we first of all you standard inv evil rate
measurements of the kind that we talked about a minute ago.
We've also used a preparation mhm.
In which the rat sciatic nerve,
the dorsal root ganglion endorsing route are
removed from the animal and
placed in a short term culture situation in vitro.
In this arrangement,
the dorsal root ganglia,
that is the cell bodies that lead to the psychotic
nerve and to the dorsal root
uh mm
are placed in a isolated chamber
separated from the media
surrounding the nerve and the roots by
silicon grease barriers,
precursors such as treated lucy and created few coast can
then be added to the ganglion chamber
and transport
uh synthesis of
proteins or black of proteins will occur here and
some fraction of those newly synthesized proteins will enter
fast transport both up the root and
down the nerve to make sure that we don't lose
transported materials.
We like it.
The roots and nerves H two
A allow quantitative evaluation
of the fraction of materials entering transport.
So this type of preparation allows measurements of
treated lucy inc,
as well as the fraction of milly synthesized materials entering
transport.
I should mention also that no transport material
or very little enters the ventral root.
As one would expect.
Uhh Now using
preparations from animals chronically
intoxicated with acrylamide compared to controls.
On the next slide,
we find no difference in total treated lucy
inc by the cell bodies or
in the fraction of newly synthesized material entering
fast transport either down the nerve or up the ventral roots
between the control and the crow might intoxicated
animals.
Similarly,
if preparations from normal animals are incubated with
acrylamide uh In the incubation media,
we find no impairment of either protein
synthesis or fast transport.
On the other hand,
measuring fast transport rates,
not the total amount that gets into the accident.
Fast transport rates we do find,
as indicated in the next slide,
some slowing a fast transport.
In this normal animal,
rapidly transported material has reached about 60
within three hours after
injection treated losing into the ventral horn.
In this acrylamide intoxicated animal,
uh about 60 millimeters has reached
four hours after injection
of translated,
losing.
So the calculated rate would be about 424
mm a day here And about
336 mm a day
here.
The next slide shows mean
values for normal and acrylamide treated
animals.
Uh And there's a slight but
significant difference between the chrome I treated animals.
Very similar results have been obtained using two five X.
And I own and other laboratories slowing a fast
transport at least Some preliminary studies has been suggested to
occur with Christine neuropathy method,
beetle ketone intoxication and try a little crystal phosphate
intoxication.
Now does this slowing of transport lead to the dyslexic
degeneration?
Yeah,
we currently feel that considerable caution is required in
making that interpretation of the data for two
reasons.
1,
with acrylamide at least the slowing of fast
transport is found only after the
initial process of excellent regeneration has
begun.
If that were the responsible factor,
you would anticipate finding a slowing of fast
transport occurring prior to the
beginning of maximum degeneration.
Also,
we have examined the role of fast
transport in another abnormality produced by
acrylamide and that's the impaired capacity of
axons to regenerate.
Following a crush acrylamide,
intoxicated nerves are not able
to grow out uh and regenerate
uh or to mature their axons as well as
normal animals has been shown by uh morgan
Hughes and by ochoa previously
and uh I am already graphic and
radiochemical studies of uh preparations
from those animals very clearly indicates
that that failure of outgrowth cannot be
ascribed to a failure of delivery
rapidly transported materials to the growing
tips.
So at present we can say that a gross impairment in cell body
protein synthesis is not requisite for the development of a
dying back picture.
It seems most likely that the slowing of fast transport at
least in the case of acrylamide.
Uh uh that the slowing of fast
transport and the dislike some of the generation are both
consequences of some as yet unknown
mechanism of action of the toxin
curly.
A major need at present is for a study
of individual proteins separated.
Uh For example,
electro theoretically.
Now,
how applicable such Studies are two.
Human degenerative diseases
is not clear.
We think that the experimental models of dying
back are quite good models of human dying back
neuropathy,
many toxic neuropathy and probably those that
occur in some degenerative conditions.
Perhaps Friedrich's ataxia.
The difficulty is that one criteria of dying back
that we previously mentioned,
the selective vulnerability of larger longer
axons can be satisfied on
clinical or pathologic grounds in human material.
With the second criteria,
the demonstration of distal to proximal
degeneration of individual actions is very difficult to
establish.
The question we might raise is whether Ailes
is a disorder in which an
element of dying back might occur.
Uh This question uh It
seems to me deserves re evaluation by classic neuropathy
logic techniques because our thinking about the
disorder uh at this point
would be colored by the extent to which it
represented a dying back process
in the extent to which an analogy to these other disorders could
be made.
But I would add that it will be very difficult to
establish the spatial temporal evolution of the
pathology in a less using classic classic
techniques.
And I'd like to mention the value of animal
models for that kind of path a biologic study.
Uh At this point there have been
no very good models of A.
L.
S.
Uh There have been
occasional models of motor neurone disease
which dr john andrews at U.
C.
L.
A.
Among others has uh studied pathologically
very extensively.
We recently have been introduced to a model uncovered by dr
linda cork who is now at Hopkins
uh of motor neurone disease.
Uh A spinal
muscular atrophy occurring in
Brittany
spaniels.
Uh These dogs,
at a few months of life develop
primarily approximately weakness and have
muscle pathology changes that are uh
strikingly similar to the human spinal muscular
atrophy changes.
Uh They of course show loss of anterior
horn cells in the spinal cord.
Uh And I would add that in addition they show
changes in accident pathology which suggests
some dynamic accidental process is going
on as well.
And this pathology includes these enormous
external swellings in the spinal cord here,
seeing an electron micrografx.
Uh So that in this situation at least
the process of motor neuron degeneration
is accompanied by an axonal degeneration that we cannot
ascribe simply to water and degeneration due to the death
of the motor neuron.
And as we've indicated before,
uh it would be of interest to
uh reevaluate um pathology
in a minute traffic lateral sclerosis with regard to the question
of whether a distal uh to proximal
degeneration occurs at any rate,
were quite interested in studying with
uh transport.
And I am already graphic techniques
these models of motor neuron disease.
Mhm.