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Cerebellar Epigenetics: Transcription
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ofmicroRNAs inPurkinje Neurons
asanApproach toNeuronal Plasticity
NealH.Barmack
19
Abstract
Cerebellar plasticity is often investigated using brief electrical coactivation of climbing and parallel ber pathways
to evoke Long Term Depression (LTD), a temporary
decrease in the excitability of Purkinje cell Simple Spikes
(SSs). The mechanistic importance of individual proteins
in climbing ber-evoked depression of simple spikes can
be approached by examining the longer-term role of regulated microRNA transcription. In this review, we examine
the consequences of longer activation (1–30h) of climbing bers on the expression of microRNAs, mRNAs and
proteins in targeted Purkinje cells of the mouse. Horizontal
optokinetic stimulation (HOKS) of unanesthetized mice
increases climbing ber discharge of occular Purkinje
cells ipsilateral to the stimulated eye. This increased
climbing ber discharge increases the expression of several microRNA and mRNA transcripts. One of these transcripts, miR335, increases 18X after 24 h of
HOKS.Pri-miR335 transcripts increase 28X after 24h of
HOKS. miR335 transcripts decay with a time constant of
~2.5h. Increases in miR335 transcripts are coupled with
decreases in the expression of calbindin, 14-3-3-θ and
protein kinase C-γ (PKC-ɣ). This interaction is critical for
the serine phosphorylation of the GABAA-ɣ2 receptor
and its membrane expression in Purkinje cells and may
contribute to homeostatic regulation of Purkinje cell discharge of SSs.
Keywords
Flocculus · Plasticity · Climbing ber
N. H. Barmack (*)
Department of Physiology and Pharmacology, Oregon Health and
Science University, Portland, OR, USA
e-mail: barmackn@ohsu.edu
19.1 Introduction
This review explores neuronal plasticity of Purkinje cells in
the cerebellar occulus. Two major afferent systems inuence the excitability of Purkinje cells: (1) Climbing bers
originate from the contralateral inferior olive. Each climbing
ber originates from the inferior olive and makes ~500 glutamatergic synapses on the dendritic tree of a Purkinje cell in
the contralateral cerebellum (Cajal 1911; Granit and Phillips
1956; Eccles etal. 1967; Konnerth etal. 1990; Harvey and
Napper 1991). Climbing bers evoke long duration (3–5ms)
“complex spikes” (CSs) that have spontaneous discharge frequencies of 0.2–0.9 imp/s (Eccles et al. 1966a; Goossens
etal. 2004; Yakhnitsa and Barmack 2006). (2) Mossy bers
originate from multiple brainstem nuclei and make synaptic
contact with a granule cell dendrite in a ‘glomerular synapse’
that includes a Golgi cell inhibitory terminal (Eccles etal.
1966b; Fox etal. 1967). Granule cell axons ascend through
the molecular layer and then divide into parallel bers that
make excitatory synapses on sagittally arrayed Purkinje cell
dendrites (Cajal 1911; Fox and Barnard 1957; Eccles etal.
1965). The summed activity of excitatory parallel ber ter-
minals and inhibitory interneurons modulates the discharge
of Purkinje cell “simple spikes” (SSs) (Granit and Phillips
1956; Eccles etal. 1965; Barmack and Yakhnitsa 2008). SSs
have a short-duration action potential (~1ms) that discharges
spontaneously at 10–30 imp/s (Goossens et al. 2004;
Yakhnitsa and Barmack 2006).
Cerebellar neuronal plasticity has often been characterized electrophysiologically by the phenomenon of ‘longterm depression’ (LTD). LTD is induced by repeated pairings
of short electrical tetanus to climbing bers in conjunction
with an electrical stimulus to a parallel ber bundle. Repeated
pairings reduce the efcacy of the parallel ber stimulus to
evoke its pre- paired response from the Purkinje cell. The
decay of LTD lasts several minutes, serving as a model for
cerebellar ‘learning.’ The occurrence of Purkinje cell depolarization evoked by climbing ber stimulation during LTD
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
D. L. Gruol et al. (eds.), Essentials of Cerebellum and Cerebellar Disorders, https://doi.org/10.1007/978-3-031-15070-8_19
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N. H. Barmack
may be substituted by direct depolarization of the recorded
Purkinje cell in conjunction with parallel ber volley (Ito
et al. 1982; Sakurai 1987; Narasimhan and Linden 1996;
Linden and Connor 2001; Crepel 2009). This seemingly simple example of neuronal plasticity, modeled by LTD, may
reect the transcription, translocation and targeting of as
many as 100 proteins (Sanes and Lichtman 1999).
LTD is impaired in mice in which the expression of protein kinase C (PKC) in Purkinje cells is reduced by a transgenic L7-Protein Kinase C Inhibitor (Goossens etal. 2001,
2004). Despite the loss of LTD, L7-PKCI mouse mutants are
still able to learn and perform sensory-motor tasks that typically reveal cerebellar impairment (Schonewille etal. 2011).
There remains no consensus on the molecular mechanisms
required to yield LTD.
Modication of sensory-motor behaviors that the cerebellum is thought to inuence, such as optokinetic modication
of the vestibuloocular reex, occur within a time course of
hours. Consequently, the longer- term molecular and biochemical consequences of sustained climbing ber activity
on functionally clustered Purkinje cells might be more amenable to experimental analysis.
A sustained discharge of occular Purkinje cells can be
controlled by horizontal optokinetic stimulation (HOKS)
(Maekawa and Simpson 1973; Simpson and Alley 1974; Alley
etal. 1975; Maekawa and Takeda 1976; Leonard etal. 1988).
Unlike natural activation of other cerebellar sensory pathways,
HOKS of occular Purkinje cells can be sustained and tolerated for up to 30h in unanesthetized mice and rabbits (Barmack
and Nelson 1987; Pettorossi etal. 1999). The subcellular consequences of prolonged HOKS can be examined subcellularly
by screening occular Purkinje cells by using assays for
microRNA (miRNA) and mRNA.miRNA screens have particular appeal because of the possibility that a single miRNA
could account for changes in the transcription of multiple protein mRNAs. These screens provide molecular clues that may
account for the subcellar changes induced in Purkinje cells by
sustained climbing ber activity and may contribute to our
understanding of cerebellar plasticity.
19.2 Biological Consequences
ofProlonged HOKS ofaClimbing
Fiber Pathway totheFlocculus
Optimal HOKS is achieved by placing a mouse at the center
of a contrast-rich sphere, that rotates about the vertical axis
at ~5 deg./s (Barmack and Hess 1980). HOKS excites
direction- selective “on” ganglion cells in the right eye while
reducing the activity of such cells in the left eye (Oyster etal.
1980) (Fig. 19.1a, b). HOKS increases CSs in occular
Purkinje cells contralateral to the eye stimulated in the
posterior→anterior (P→A) direction. If HOKS is maintained
for more than 1 h it evokes a prolonged optokinetic afternystagmus that lasts for several hours (Barmack and Nelson
1987; Pettorossi et al. 1999). Following HOKS the stimu-
lated and control occuli can be removed and prepared for
subcellular analysis using microarrays for mRNA and
microRNA (miRNA) (Fig. 19.1a, b). Ganglion cell axons
from the right eye project to the left nucleus of the optic tract
(NOT) in the dorsal midbrain. The axons of neurons in the
left NOT descend to the inferior olive where they excite neurons in the left dorsal cap (DC) of the left inferior olive
(Maekawa and Simpson 1973; Alley et al. 1975; Barmack
and Hess 1980; Simpson etal. 1988). Neurons in the left dorsal cap project as climbing bers to the right occulus where
they excite Purkinje cells (Leonard etal. 1988; Schonewille
etal. 2006). Since the visual projections to the occulus are
lateralized, HOKS of the right eye in the P→A direction for
0–24h increases climbing ber discharge in the right occulus while decreasing climbing ber discharge in the left
occulus. HOKS can be maintained for 0–30h.
19 Cerebellar Epigenetics: Transcription ofmicroRNAs inPurkinje Neurons asanApproach toNeuronal Plasticity
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Fig. 19.1 Horizontal optokinetic stimulation (HOKS) evokes climbing
ber-induced increases in microRNA transcription in the occulus. (a)
Cartoon depicts an optokinetic pathway from the retina to the ipsilateral
occulus (see text for details). The dashed lines indicate the excitatory
pathway that originates from the right eye during counter-clockwise
HOKS.The solid lines indicate the “disfacilitated” pathway caused by
HOKS of the left eye. (b) Mice are restrained at the center of an optokinetic sphere that rotates CCW at 6deg./s and excites ganglion cells in
the right eye. Mice receive binocular HOKS for xed durations of
0–30h. (c) The left and right occuli are dissected and RNA extracted.
cDNAs are synthesized and amplied by PCR.The U6 promoter is coamplied as a loading control. Each reaction is run on a gel and the
optical density of the bands is measured photometrically. When the
19.3 Transcription ofmiRNAs inPurkinje
Cells
The regulation of gene transcription, translation and targeting of proteins could be simplied if the proteins are regulated by common precursors such as microRNAs; small,
non-coding RNAs derived from “junk” DNA. A single
microRNA could target the 3′-untranslated regions of as
many as 5–30 mRNAs and limit their translation by complementary repression and degradation (Cullen 2004; Robins
and Press 2005; Landgraf etal. 2007; Hobert 2008; Eulalio
etal. 2009; Guo etal. 2010). The transcription of microRNAs has been linked to cellular development, and apoptosis
ratio of PCR band density (R.Floc./L.Floc.) is >1 it indicates increased
transcription of miR335 in the right occulus. Two microRNAs,
miR125 or miR147 (gray bars), are run as controls. HOKS duration is
indicated above each gel pair and for each histogram bar. (d) Transcripts
of miR335in the right occulus decay rapidly. The transcripts are measured at the indicated times after HOKS is stopped. (e) A digoxigeninlabeled oligonucleotide complementary to miR335 and immunolabeled
with an antibody to digoxigenin hybridizes in the cytoplasm of Purkinje
cells. The area denoted by the small box is shown at a higher magnication in the larger insert. DC dorsal cap of the inferior olive, Fl occulus,
NOT the nucleus of the optic tract, vPFl ventral paraocculus. [Modied
from (Barmack etal. 2010)]
(Reinhart et al. 2000; Ambros 2004; Cullen 2004; Harfe
2005; Kosik 2006; Schratt etal. 2006; Bushati and Cohen
2007), oncogenesis (Hayes etal. 2014), epilepsy (Alshara
et al. 2015) and microbial defense (Bartel 2004; Cullen
2004; Zeng etal. 2005). microRNAs also regulate the func-
tions of adult neurons (Schratt 2009; Smalheiser and Lugli
2009; Barmack et al. 2010; Konopka et al. 2011; Mellios
etal. 2011; Tognini etal. 2011).
After HOKS stops, mice are anesthetized, the left and right
occuli are removed and total RNA is extracted from each occulus. cDNAs are synthesized and amplied for initial analysis
by a microarray (GeneChip® microRNA 2.0, Affymetrix Co).
Three miRNAs (miR126, miR335 and miR379) are differen-
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N. H. Barmack
tially expressed in stimulated occuli stimulated by HOKS
relative to contralateral controls. The positive identication of
specic miRNAs is conrmed by subsequent measurements
using specic primer pairs with greater accuracy. These conrming tests use either PCR amplication and northern blot
gels or polymerase chain reaction (qPCR).
19.4 HOKS Increases miR335 Transcripts
andthis Increase Decays Rapidly
Depending onHOKS Duration
The minimal duration of HOKS necessary to evoke a detectable increase in microRNA transcripts is measured by experiments in which mice receive HOKS for different durations
(0, 3, 6, 12, 24, 30h) before they are euthanized. Increases in
miR335 transcripts can be detected after 6 h of
HOKS.Transcripts of miR335 continue to increase linearly
from 6 to 30h (Fig.19.1c).
The increase in miR335 transcripts evoked by HOKS rapidly decreases when HOKS stops. This question is addressed
specically by exposing mice to a constant duration of
HOKS for 24h. The HOKS then stops and the mice remain
within the illuminated sphere for 0.0, 1.5, 3.0, 6.0, or 12h
before they are euthanized. Transcripts of miR335 are then
measured at each of the specied post-stimulus intervals.
miR335 transcripts decay to control levels with a time constant of ~2.5h (Fig.19.1d). This rapid decay suggests that
miR335 transcripts cannot account singularly for longlasting changes in Purkinje neuronal excitability.
19.5 Hybridization Histochemistry
Localizes miRNA Transcripts
toPurkinje Cells
While HOKS increases microRNA transcripts in the occulus, it is by no means certain that the transcripts are localized
exclusively to Purkinje cells. This is tested using “locked
nucleic acid-modied oligonucleotide probes” to examine
whether they hybridize with mature microRNAs. A probe for
miR335 hybridizes with Purkinje cell soma, but not with
Purkinje cell nuclei (Fig.19.1e). This conrms the cytoplasmic location of the mature miR335in Purkinje cells. Other
probes for microRNA transcripts that increase with HOKS,
miR15, miR21 and miR361, also hybridize with Purkinje
cells and weakly with stellate cells. A scrambled probe, having the same GC content as the probe for miR335, fails to
hybridize with either Purkinje cells or other cerebellar neurons. These data conrm that microRNAs evoked by climbing ber activity are localized to Purkinje cells.
19.6 Central Floccular Zone is
aHomogeneous Source ofPurkinje
Cells Excited by HOKS
The mouse occulus comprises a single spindle-shaped
folium with an axial length of 1.1mm. The linear extent of
the occular folium is ~400 μm at its peak. It weighs
~400μg. The climbing bers that are activated by HOKS
project to the middle third of the occulus. Climbing ber
zones are established by electrophysiological recordings
from Purkinje cells during optokinetic stimulation
(Goossens et al. 2004). Such recordings reveal a central
zone in which the climbing ber responses of Purkinje cells
are modulated by P→A HOKS of the ipsilateral eye. This
central zone is anked by two zones in which Purkinje cell
activity is modulated by vertical optokinetic stimulation
(VOKS). Purkinje cells in the most caudal occular C2
zone are unresponsive to optokinetic stimulation and may
be linked to the control of head movement (De Zeeuw and
Koekkoek 1997). Excising the middle third of the occulus
increases the concentration of Purkinje cells in the sample
that are excited by HOKS (Fig.19.2a). After 24h of HOKS
miR335 transcripts from this central zone (red) are 3X
more abundant than in previous experiments where transcripts in the total right occulus were compared to transcripts in the total left occulus (Figs. 19.1c vs 19.2a).
HOKS does not inuence miR335 transcripts in anking
occular zones. In these zones the right/left occulus ratio
is ~1.
Floc. climbing fiber zones
19 Cerebellar Epigenetics: Transcription ofmicroRNAs inPurkinje Neurons asanApproach toNeuronal Plasticity
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ab c
1,200
4
rostral
24
16
N = 4
8
HOKS-evoked miR335
R.Floc./ L.Floc.
0
Rostral Middle
600
3
1
2
C2
caudal
0 µm
1 mm
*
Caudal
35
30
25
20
15
10
HOKS increases pri- and mature miR335
R.Floc./ L.Floc.
5
0
Floc. control
Mature miR335
Pri-miR335
Pri-miR335
Mature miR335
N = 6
*
Total Floc.
*
AAA
*
*
Middle Floc.
Pull-down of PKC-γ by
de f
2.5
2.0
*
miR335
14-3-3-θ mRNA
14-3-3-ε mRNA
*
PKC-α
His-tagged 14-3-3-θ
Lysate
Beads
1 2 3
His-14-3-3-θ
HOKS modulates 14-3-3-θ
mRNA transcription
1.0
*
0.5
14-3-3 transcripts, R.Floc./L.Floc.
0.0
PKC-γ
η
θ
γ
ε
σ
ζ
Immunoprecipitation of
14-3-3-θ and PKC-γ
Lysate
14-3-3-θ IP
1 2
1.5
PKC-δ
1.0
PKC-β
0.5
Transcripts in N2a cells
re: U6 controls
*
0.0
miR335 inhibitor
Fig. 19.2 HOKS-increases pri-miR335 and miR335 and increases
their interaction with proteins. (a) Purkinje cells removed from the horizontal occular zone contain 3X more concentrated miR335 transcripts
than do Purkinje cells removed from the total occulus following 24h
of HOKS. miR335 transcripts in the anking occular regions are not
increased by HOKS. (b) The relative number of transcripts of both primiR335 and mature miR335 are measured to determine if the HOKSevoked mi335 transcripts could be attributed to increased transcription
of miR335 or perhaps decreased degradation of constitutive transcription of miR335. Floccular samples from the right central occular are
compared with samples from the entire left occulus. HOKS evokes
larger increases in pri-miR335 transcripts than mature miR335 transcripts. (c) HOKS for 24h decreases 14-3-3-θ transcripts of the occulus onto which HOKS-excited climbing bers project. mRNA
transcripts for only one 14-3-3 isoform, 14-3-3-θ, decrease (ANOVA,
p<0.04, N=3). (d) Interaction of miR335 with14-3-3-θ mRNA can be
examined more directly invitro using N2a cells in which transcripts of
both miR335 and14-3-3-θ mRNA are native. N2a cells are infected
with an miR335 precursor or an miR335 inhibitor. Transfection of N2a
cells with the miR335 inhibitor decreases transcripts of miR335 and
increases transcripts of 14-3-3-θ. Conversely, transfection of N2a cells
*
PKC-γ
miR335 precursor
Lysate
14-3-3-θ
with an miR335 precursor increases transcripts of miR335 and
decreases transcripts of 14-3-3-θ. The selectivity of these interactions is
supported by the absence of changes in transcripts of the control isoform, 14-3-3-ε. Transcripts for U6 serve as a loading control for
miR335. Transcripts for actin served as a loading control for 14-3-3-θ
and 14-3-3-ε. (e) 14-3-3-θ interacts with PKC-γ in cerebellar lysates.
Cerebellar lysates extracted from three mice and incubated with recombinant 14-3-3-θ
beads. The beads are mixed with cerebellar lysates to test for interactions of 14-3-3-θ with other proteins. In Lane 1 cerebellar lysates are
immunolabeled with antibodies to PKC isoforms (PKC-α, PKC-δ,
PKC-β and PKC-γ). Lane 2, shows the absence of non-specic binding
to his-binding beads without 14-3-3-θ attached in cerebellar lysates.
Lane 3 shows specic binding of 14-3-3-θ to PKC-γ. (f) The interaction
of PKC-γ with 14-3-3-θ
cerebellar lysates with antibodies to either 14-3-3-θ or PKC-γ. After
re-suspension the immunoprecipitated lysates test positive with antibodies to both 14-3-3-θ and PKC-γ, indicating that they interact invivo.
[Modied from (Schonewille etal. 2006; Barmack etal. 2010; Qian
etal. 2012)]
probes are attached to nickel- impregnated agarose
is conrmed by co- immunoprecipitation from
PKC-γ IP
1 2
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N. H. Barmack
19.7 HOKS Evokes More Pri-miR335
Transcripts than miR335 Transcripts
Proof of climbing ber-evoked increases in microRNA transcripts is not necessarily proof of increased microRNA transcription. Several enzymatic post-transcriptional factors could
contribute to the regulation of microRNAs. However, the transcription of larger pri-microRNAs precedes the action of intranuclear enzyme Drosha/Pasha that digests pri-microRNA
converting it into pre-microRNA (Lee etal. 2003; Cai etal.
2004). It also precedes the action of cytoplasmic enzymes
such as Dicer, that cut pre-miRNA into a microRNA duplex
(Lund and Dahlberg 2006) and Argonaut, which selects the
mature miRNA strand from the miRNA duplex (Ghildiyal and
Zamore 2009; Johnston and Hutvagner 2011). If climbing
ber-evoked depolarization of Purkinje cells increases mature
miRNA transcripts by rst increasing transcription of primiRNAs rather than by post- transcriptional regulatory mechanisms, then increased transcripts of both pri-miRNAs and
mature miRNAs should be detected after HOKS-evoked
climbing ber excitation of Purkinje cells. This question can
be addressed by nding primer binding sites in pri-miR335
that are not present in mature miR335 (Fig.19.2b).
Following 24 h of HOKS, the right occulus can be
divided into thirds once again to increase the likelihood of
maximizing the recovery of miR335 transcripts. The concentration of pri-mR335 transcripts recovered from the middle
zone of the stimulated (right) occulus is 28X greater than
the concentration of transcripts recovered from the unstimulated left occulus. Mature miR335 transcripts are 18X more
concentrated. When this experiment is repeated using samples taken from the whole right occulus, pri-miR335 transcripts are 9X more concentrated in the right occulus and
mature miR335 transcripts are 5X more concentrated. In
control samples taken from mice not exposed to HOKS, the
ratio of right occulus/left occulus for pri-miR335 and
mature miR335 is 1. The increased concentration of miR335
transcripts evoked by HOKS can be attributed to increased
transcription.
The 28X increase of pri-miR335 transcripts in the middle
zone of the right occulus exceeded the 18X increase in
miR335 transcripts. Possibly this difference can be attributed
to their different cellular locations. pri-miRNAs are conned
to the nucleus where they are converted by a microprocessor
complex into pre-microRNA that is exported to the cytoplasm and converted to miRNAs (O'Brien etal. 2018).
repressed by miR335. This can be accomplished using multiple screens for identity. First, the nucleotide sequence of
miR335 offers a clue to the identity of target mRNAs that
have complementary sequences. A stringent screening of
two databases (microRNA Registry and EnsEMBL) reveals
more than 149 mRNAs with sequence complementarity to
miR335. This is unacceptably large. Second, a more direct
empirical approach uses an mRNA array to measure reductions in transcripts of potentially targeted mRNAs recovered
from the occulus, again following 24h of binocular HOKS
(Genome 430 2.0, Affymetrix). Such an mRNA screen
reveals a total of 42 mRNA transcripts that decrease after
24 h of HOKS (Barmack and Qian 2002; Barmack et al.
2010). This mRNA screen cannot distinguish between
mRNA reductions that can be attributed to miRNA repression specic to miR335, repression due to other miRNAs or
repression due to other molecular interactions. Third, one
can microinject specic miR335 inhibitors directly into the
cerebellum and identify mRNA transcripts that increase as a
consequence of the microinjection, again using an mRNA
microarray. This approach generates 28 mRNAs whose transcripts increase following the microinjection of miR335
inhibitors. Two mRNAs satisfy each of the three screens. The
mRNAs for 14-3-3-θ and calbindin are predicted to interact
with miR335 based on sequence complementarity. They are
repressed in vivo when the occulus concentration of
miR335 transcripts is elevated by HOKS and their concentration in the occulus increases when an miR335 inhibitor
is microinjected into the occulus (Barmack et al. 2010,
2014).
19.9 Identication of14-3-3 Isoforms
inMouse Flocculus
The 14-3-3 family of seven isomers play a regulatory role in
signal transduction and by binding to partner proteins. Six of
seven 14-3-3 isoform mRNAs (θ, η, ε, γ, ζ, β and σ) are
detected in the cerebellum (Fig.19.2c). The levels of transcripts of each of these isoforms in the occulus reveals
whether their expression is inuenced by HOKS.Only transcripts for 14-3-3-θ decrease in the right occulus following
24h of HOKS in the P→A direction of the right eye.
19.10 miR335 Represses 14-3-3-θ mRNA
inN2a Cells
19.8 Screens forDiscovering mRNAs
Targeted by microRNAs
Having established that miR335 transcription in Purkinje
cells increases targeted by prolonged climbing ber activity,
it is useful to identify the proteins whose translation is
Increased climbing ber activity increases transcripts of
miR335 and decreases transcripts of calbindin and 14-3-3-θ
mRNAs. However, the decrease in mRNAs may be inuenced by other subcellular factors related to prolonged
climbing ber-induced discharge rather than by miR335
repression of calbindin and 14-3-3-θ mRNAs. The interac-
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tion of miR335 with its target mRNAs can be studied more
directly invitro using N2a cells in which miR335, calbindin
and 14-3-3-θ mRNA transcripts are expressed naturally. The
consequences of manipulating the concentration miR335in
N2a cells on the concentration calbindin and 14-3-3-θ
mRNA transcripts can be measured directly by transfecting
N2a cells with either an miR335 inhibitor or an miR335 precursor (Fig.19.2d). Twenty-four hours after the N2a cells are
transfected with either an miR335 inhibitor or an miR335
precursor they are harvested and transcripts for miR335,
14-3-3-θ and 14-3-3-ε (control) are measured using
qPCR.Transfection of N2a cells with an miR335 inhibitor,
decreases transcripts of miR335 and increases transcripts of
14-3-3-θ. Conversely, transfection of N2a cells with an
miR335 precursor increases transcripts of miR335 and
decreases transcripts of 14-3-3-θ. The selectivity of these
interactions is supported by the absence of changes in transcripts of the control isoform, 14–3-3-ε, that does not interact with miR335.
19.11 14-3-3-θ Interacts withPKC-γ
intheCerebellum
Functioning as part of a sequence of steps in the cell cycle,
proteins bind with and stimulate PKC-ε activity controlling
the nal step in cell division (Saurin etal. 2008). Possible
interactions between 14-3-3-θ and PKC-γ can be studied
using a plasmid in which a histidine-tagged 14-3-3-θ is
attached to nickel-impregnated agarose beads. These
histidine- tagged 14-3-3-θ beads can be used identify proteins in cerebellar lysates with which they interact. The
histidine- tagged beads along with their attached proteins can
be separated from other proteins in a cerebellar lysate by
centrifugation, resuspended and immunoblotted antibodies
to PKC isomers (PKC-α, -β, -γ and -δ) that are expressed in
the cerebellum (Fig.19.2f). 14-3-3-θ interacts with PKC-γ,
but not other PKC isoforms (α, β and δ). The specicity of
this interaction is conrmed by the use of antibodies to 14-33-θ and PKC-γ to co-immunprecipitate associated proteins
from a mouse cerebellar lysate. An antibody to 14-3-3-θ coimmunoprecipitates PKC-γ. An antibody PKC-γ coimmunoprecipitates 14–3-3-θ. These data indicate that
PKC-γ and 14-3-3-θ interact in cerebellar lysates and promote serine phosphorylation and cell–surface expression the
GABAA-ɣ2 receptors (Qian etal. 2012).
19.12 miRNAs andCerebellar Plasticity
microRNAs account for some of the neuronal interactions
that occur in Purkinje cells following HOKS.Several observations highlight this possibility: First, occular Purkinje
cell miRNA transcripts increase with the duration of HOKS
for up to 30h. Second, the increase in microRNA transcripts
is maintained for 2.5 h after HOKS is stopped. Third, the
HOKS-evoked increase in climbing ber activity increases
miR335 transcription in Purkinje cells, but not cerebellar
interneurons. This increased transcription of miR335 (18X)
follows an even larger increase (28X) in pri-miR335 transcripts. Fourth, in both in vivo and in vitro experiments
changes in microRNA expression inuence protein expression. These data suggest that miRNAs may initiate changes
in neuronal function, but do not account for maintained
changes.
The value of this experimental approach lies not in the
discovery of every miRNA whose transcription is related to
climbing ber activity. Rather it focuses attention on a few
clusters of miRNAs that co-regulate protein translation during the activity of multiple neural systems that respond to
glutamatergic transmission. This may lead to a better understanding of the subcellular changes that account for neuronal
plasticity. It may also lead to prospective pharmacological
targets to suppress the consequences of the abnormal glutamatergic activity.
An understanding of the interactions between microRNAs and mRNAs may prove useful in treating cerebellar disorders. Already microRNA dysfunction has been linked to
neurological diseases such as cerebellar ataxia (Schaefer
et al. 2007; Barnes et al. 2011), spinal muscular atrophy
(Haramati etal. 2010) and polyglutamine-induced neurodegeneration (Bilen et al. 2006). Pharmacological treatments
that target specic microRNAs or proteins, whose translation
is repressed by microRNAs may provide novel therapeutic
approaches for the treatment of aberrant neurological and
oncological disease.
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The Genetic Programs Behind
https://t.me/medicina_free
Cerebellar Development
KathleenJ.Millen
20
Abstract
Careful analyses of cerebellar phenotypes of mice with
single gene mutations have been central to dening the
developmental programs that build the cerebellum. These
studies have revealed the complex and dynamic intracellular and extracellular mechanisms driving the formation
of this stereotypically laminated and foliated structure.
They have also provided genetic tools to manipulate
genes and cells to further probe cerebellar development
and function. The eld continues to evolve. Large-scale
transcriptomic and genomic analyses are revealing master
regulatory cascades. Work from non-traditional model
organisms and developing human cerebellum are providing new insights which impact our understanding of neurodevelopmental disorders involving the cerebellum.
Keywords
Neurodevelopment · Malformation · Patterning
20.1 Introduction
There is long-standing and continually growing evidence
that the cerebellum has extensive and central roles in numerous major brain functions beyond its stereotypically appreciated role in motor control. For example, fMRI studies show
just 20% of the human cerebellum is dedicated to areas
involved in physical motion. The other 80% is broadly connected to brain regions regulating abstract thinking, planning, emotion, and memory (Marek etal. 2018). Clinical and
model organism studies demonstrate roles in social and lan-
K. J. Millen (*)
Center for Integrative Brain Research, Seattle Children’s Research
Institute, Seattle, WA, USA
Department of Pediatrics, University of Washington School of
Medicine, Seattle, WA, USA
e-mail: kathleen.millen@seattlechildrens.org
guage and even more recently, satiety (Low et al. 2021;
Metoki etal. 2022; Schmahmann 2019). These functions are
based on circuits embedded in the magnicently foliated and
laminated mature cerebellar structure that emerges as a result
of highly regulated and integrated, genetically driven developmental processes. Hence a solid understanding of cerebellar development can be central to understanding the
underpinnings of cerebellar disorders including both developmental disorders and adult neurodegenerative disorders.
However, understanding cerebellar development means
understanding the origins of cerebellar cells, the considerable cellular migrations and dynamic morphological changes
that occur to build the mature cerebellar structure.
Numerous reviews are available describing the major
developmental events that build the mature cerebellum
(Chizhikov and Millen 2020; Haldipur and Millen 2019;
Leto etal. 2016; Lowenstein etal. 2022). Here I will simply
provide an overview to contextualize the rest of the discussion in this chapter (Fig.20.1). The cerebellum is a derivative
of dorsal rhombomere 1, the segment of the anterior hindbrain, just posterior to the mid-hindbrain junction. This junction is central to the development of the cerebellum, forming
the Isthmic Organizer, a morphogen-secreting signaling center that patterns and denes the boundaries of the cerebellar
territory in the early neural tube. All cerebellar neurons
derive from two primary progenitor zones: the ventricular
zone (VZ) adjacent to the dorsal fourth ventricle, and the
rhombic lip (RL), located dorsally between the roof plate
and the ventricular zone. The roof plate, located at the top of
the neural tube is essential to pattern the RL.Overlapping
waves of neurogenesis from the VZ give rise to the
GABAergic neurons of the cerebellum, rst generating
GABAergic cerebellar nuclei neurons, Purkinje cells, then
the GABAergic interneurons (Lowenstein et al. 2022) as
cerebellar lamination matures. As VZ neurogenesis proceeds
and differentiating Purkinje cells migrate outwards along
radial glial bers to form a Purkinje cell plate at the periphery of the now bulging cerebellar anlage, the 2 paired wings
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
D. L. Gruol et al. (eds.), Essentials of Cerebellum and Cerebellar Disorders, https://doi.org/10.1007/978-3-031-15070-8_20
137
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