1. The dendritic morphology of wild-type and NRN knockdown striatal
MSNs was studied by using the culture system. At DIV2, the cells on
half of the cover slips were infected with an shRNA Scrambled (shsc)
control, and NRN in the knockdown group was silenced with a neuritin
shRNA expressing virus. Since the virus also expressed the gfp gene, the
infected cells were identified with an EGFP fluorescence. The cells were
also stained for DARPP32 (D32), a regulatory protein found in MSNs,
to identify MSNs. Four different time points were chosen for fixation
and staining in order to gauge the spine development pattern for normal
MSNs, and also to see if the effects of neuritin vary over time in MSN
development. Our timeline is shown in Figure 1.
Analysis was focused on dendritic spines. The density of spines on
secondary dendrites of GFP/D32 double-stained cells was determined in
order to quantify the results, for both control and knockdown groups. By
separating the dendritic spines into two groups—mature and immature—
we also quantified differences in spine maturation between the wild-type
and knockdown neurons. Mature spines were characterized by a wide
“mushroom-like” head measuring at least twice as wide as its stubby
base. Any visible protrusions that were less than 5 μm in length and did
not fit the mature criteria were counted as immature spines. Anything
longer than 5 μm was considered a branch and therefore was not
counted.
The role of neuritin—a synapse-maturing
protein—in striatal dendritic morphology
Anastasiya Vasilyeva, Akiko Terauchi, Hisashi Umemori
Boston Children's Hospital, Kirby Neurobiology Center, Boston, Massachusetts
Introduction
Discussion
Objective
Methods
Synapses are the sites where information processing occurs in the brain.
They consist of a presynaptic terminal, which originates from the axon
of the presynaptic cell, and a postsynaptic terminal, originating from the
dendrite of the postsynaptic cell. Since improper synapse formation and
function results in numerous neurological diseases, by studying the
molecular underpinnings of synapse formation, contributions to
treatment and preventions of such diseases can be made. For instance,
the striatum has been implicated in Parkinson’s disease, schizophrenia,
and addiction. 90-95% of striatal neurons are medium spiny neurons
(MSNs) [1]. Mature MSNs have dendritic spines, small protrusions from
the dendrite that directly receive input from the axon of the presynaptic
cell. To understand the mechanisms of synapse formation in the striatum,
the involvement of candidate molecules in the spine development of
MSNs was investigated. Neuritin (NRN), a glycosylphoshatidylinositol-
anchored protein, became the focus because it has been shown to
promote dendritic growth and synapse formation in other brain
structures, such as the hippocampus and the cortex, [2-3] and is highly
expressed in the striatum.
To investigate whether and how neuritin contributes to spine
morphology in striatal medium-spiny neurons.
No significant differences in the spine densities or maturation of wild-type and neuritin knockdown MSNs were found. However, a useful trend of spine growth in MSNs
could be seen, as shown in Figure 3. Spines were observable at DIV11 and the density rapidly increased at DIV19, while the number of mature spines increased gradually
throughout the observed time period.
Our results show that NRN knockdown does not have any apparent effect
on MSNs within the 19 days in vitro that was studied. This could indicate
that NRN’s role in synapse formation might exist exclusively in brain
structures such as the hippocampus and the cortex, and not in the striatum.
Alternatively, it is possible that since striatal NRN expression persists into
adulthood, NRN might become important after DIV19 for striatal synapse
stabilization or maintenance in adults—this point requires further
investigation.
References
1. Matamales, M., Bertran-Gonzalez, J., Salomon, L., Degos, B., Deniau,
J., Valjent, E., . . . Girault, J. (2009). Striatal Medium-Sized Spiny
Neurons: Identification by Nuclear Staining and Study of Neuronal
Subpopulations in BAC Transgenic Mice. PLoS ONE, 4(3).
2. Nedivi, E. (1998). Promotion of Dendritic Growth by CPG15, an
Activity-Induced Signaling Molecule. Science, 1863-1866.
3. Fujino, T., Leslie, J., Eavri, R., Chen, J., Lin, W., Flanders, G., . . .
Nedivi, E. (2011). CPG15 regulates synapse stability in the developing
and adult brain. GENES & DEVELOPMENT, 2674-2685.
Figure 1: Timeline for infection and staining of striatal cells in vitro.
This figure shows the experimental timeline for shsc and shNRN virus infection at
DIV2, as well as for fixation and staining at DIV11-19. This period was examined
in order to assess the pattern for spine development in striatal MSNs .
Figure 3: Spine density of immature and mature spines in wild-type and NRN
knockdown neurons in vitro.
Spine density was calculated using measurements from ImageJ software of neuron
images of DIV11-19 stained MSNs. a) Spine density between the two groups was
compared, with no significant differences. b) A gradual increase in overall spine
maturation was observed within the DIV11-19 time period, as well as a spike in
overall spine density around DIV19.
a)
b)
DIV14
controlcontrolcontrol
GFP
GFP
shNRNshNRNshNRN
D32
D32
merged
merged
DIV17
GFP
GFP
control control control
D32
D32
merged
shNRN shNRN
merged
shNRN
controlcontrolcontrol
DIV19
GFP
GFP
shNRNshNRNshNRN
D32
D32
merged
merged
Figure 2: Representative images of GFP/D32 double-stained MSNs at DIV11-19.
Images were taken of secondary dendrites from GFP/D32 double-stained neurons in good growth conditions, where the cells were surrounded by a number of other neurons,
but were sparse enough to photograph with a confocal microscope. There was a noticeable increase in spine density on DIV19.
mature
mature
DIV11
GFP
GFP
D32
D32
merged
merged
control
shNRN
control control
shNRN shNRN
immature
Results
Results
DIV0 DIV19DIV14 DIV17DIV11DIV2
virus infection
Methods
(Hering et al. 2001)
(Wang et al. 1999)
MSNs 96% GABA 2% TANs 2%Striatal
Neurons:
(Moghaddam
et al. 2015)
immature
mature