VETS 1032 Cytology Group Assignment

Word count (excluding image captions and references): 1770

Introduction to the disease

Neuronal ceroid lipofuscinosis (NCL) is a group of neurodegenerative
disorders that affect the brain and central nervous system through the
accumulation of lipopigments, fats and proteins, in the body tissues and
cells. The forms of NCL are differentiated by the age of onset, duration
and early symptoms (Goebel, Mole & Lake, 2016). The different types of
NCL are increasingly being referred to collectively as Batten Disease and
are autosomal recessively inherited (National Institute of Neurological
Disorders and Strokes, 2016). Characteristics of NCL include vision
impairment, seizures, small heads, progressive loss of nerve cells in the
brain and permanent and progressive impairment in motor and
psychological ability (Genetics Home Reference, 2016).
Animal models, naturally occurring in sheep and dogs, as well as
genetically-engineered mice, yeast, fish and some insects, are being used
to study the function of NCL proteins because it appears that many of the
NCL genes function similarly as in humans (National Institute of
Neurological Disorders and Strokes, 2016). For example, in the sheep
model, brain atrophy, vision degeneration and increase of lipopigments,
particularly a protein known as subunit c, were found to resemble the
human manifestation of NCL (Goebel, Mole & Lake, 2016).

Description of the image

Figure 1: Image to be analysed, for which the sample is known to have

been taken from the cerebellum of an animal suffering from Nuclear

Ceroid Lipofuscinosis (NCL). Black scale bars represent 20m, while the
white scale bar in the inset represents 250nm (Abitbol, 2010).
Of the two slides shown above, the left is non-diseased and the right is the
diseased. There is an obvious difference in the composition of the two
slides, the normal tissue showing orderly layering and segregation of cells.
From the spongy pink matter in the upper left corner to the diagonal line
of circular shaped cells of a deeper pink and lastly to the dense purple
granulate material in the bottom right hand corner. The diseased tissue
has none of this order and fewer of the circular shaped cells. This shows
that the disease seems to disrupt the organisation of these organelles.

Analysis of the image

The scale of images C and D (Figure 1), as well as the colour, indicate that
the images were obtained from light microscopy, as light microscopy
magnifies images only by up to 400 times (i.e. a structure that appears to
be 1cm in the ocular at highest magnification is actually 25m). The inset
in Figure 1D, which has a much smaller scale, is in greyscale, and
represents a 2D image, indicating that it was obtained by Transmission
Electron Microscopy (TEM).
At this scale, which is the approximate scale of the cell, the dark circle of
membrane with a dark purple circle in the centre, of about 8m in
diameter, resembles the structure of the nucleus, which suggests that the
sample was stained with hematoxylin (Fischer, et al., 2008).
By comparison to a normal histological section of the cerebellum, stained
with HE (hematoxylin and eosin) stain (Figure 2), it is evident that in Figure
1C, the less cell-dense area is the molecular layer, containing both the
Purkinje cell dendrites and the granule cell parallel fibres, and a few nuclei
(dark purple circles) belonging to either stellate or basket cells; the large
membrane-bound structures of 20m in diameter are Purkinje cells, which
form a single layer; and the densely-packed layer of cells is the granule
layer, containing many granule cells (Figure 3). Figure 1C therefore seems
to show normal cerebellar cortex tissue.
Figure 1D shows the same structures as Figure 1C, with the exception that
the Purkinje cells contain pink granular material within their cytoplasm,
with none of the other cells seeming to be affected. The granular cell layer
is also uneven, and Purkinje cells are abnormally-shaped and less
numerous, suggesting degradation and loss of Purkinje cells. The inset
shows a magnification of a large pink granule within these, which has a
concentric membranous structure and contains one small circular
structure. By comparison to an image of an inclusion in a Purkinje cell of a
Daschund affected by NCL (Figure 5), which was identified by the authors
to be lipofuscin (Vandevelde & Fatzer, 1980), it can be deduced that this
granular material is therefore lipofuscin which has accumulated within the
Purkinje cells in Figure 1D. The circular inclusion can also be identified as a
lipid droplet, by comparison to the structure of lipofuscin in Purkinje cells
of dwarf lemurs (Gilissen & Staneva-Dobrovski, 2013).

Lipofuscin usually appears brown under the light microscope, but stains
pink with Periodic Acid-Schiff stain, since lipofuscin is composed of crosslinked protein residues (Brunk & Terman, 2002) and protein and
carbohydrate is stained pink with PAS (Pernik, 2015). The full stain for
these images is therefore PAS and hematoxylin.
Accumulation of lipofuscin within the cell is a key symptom of NCL, where
the dysfunction of sphingolipid metabolism causes accumulation of
lipofuscin (Persaud-Sawin, et al., 2007). Lipofuscin further interferes with
homeostasis of the cell and eventually causes degradation and then
apoptosis of Purkinje cells (Abitbol, 2010), indicating that the Purkinje cells
in Figure 1D are dying. While several different strains of the disease exist
across species and breeds, neither the ultrastructure of the lipofuscin nor
the histological appearance of the disease can be used to reliably identify
the particular strain (Vandevelde & Fatzer, 1980). It can therefore be
concluded that NCL in American Staffordshire Terriers is characterised by
abnormal accumulation of membranous lipofuscin within the cytoplasm of
Purkinje cells in the cerebellar cortex, which causes their degradation and
loss.

Figure 2: Histological section of human cerebellar cortex, viewed under a

light microscope and stained with HE (Indiana, 2008).

Figure 3: Annotated version of Figure 1C, based on the conclusions of the

analysis made in the previous paragraph.

Figure 4: Annotated version of Figure 1D, based on the analysis in the

previous paragraph.

Figure 5: Ultrastructure of lipofuscin in Daschund Purkinje cells. The

arrows identify visible membrane stacks. Obtained by TEM (Vandevelde &
Fatzer, 1980).

Figure 6: Electron micrograph of lipofuscin in the Purkinje cells of a 15year-old dwarf lemur. Lipid droplets are identified by the letters 'Li', while
pigment granules are identified by a white arrow. Obtained by TEM,
87,250x magnification (Gilissen & Staneva-Dobrovski, 2013).

First cellular structure/organelle

The nucleus of the cell is usually located in the centre of the cell. The
shape and size of the nucleus varies accordingly to the cell type and the
morphology of the cell. The nucleus is enclosed by a nuclear envelope - a
double concentric layer nuclear membrane with multiple nuclear pores,
the layers being separated by a perinuclear space. The nuclear envelope
acts as a physical barrier to separate the nucleoplasm from the
cytoplasm. Nuclear pores help to regulate the exchange of

macromolecules between the nucleoplasm and the cytoplasm. On the

outer membrane of the nucleus, ribosomes are attached on its surface as
it is continuous with the membrane of the rough endoplasmic reticulum
(RER), while the inner membrane of the nuclear envelop is bound to the
nuclear lamina which supports the nucleus. The nuclear lamina is
attached to the chromatin threads in the nucleoplasm, where the heredity
materials of the cells are stored in the form of deoxyribonucleic acid
(DNA). As genes are able to be turned off and on, the nucleus is able to
regulate gene expression. Similarly, the nucleolus is also located in the
centre of the nucleoplasm. It is a dense chromatin structure which is
essential for the synthesis activity of the cells such as ribosomes
production, RNA and proteins synthesis.
Organelles which the disease affects
Neuronal ceroid lipofuscinosis, being a lysosomal storage disorder, leads
to the intracellular defects mainly targeting the lysosomes within cells,
however effects of the disorder are also able to be seen in the
endoplasmic reticulum of the cell (Jalanko & Braulke, 2009). Generally, the
main effect of the disorder is that lysosomes, and possibly other
organelles, will begin to accumulate lipopigments known as lipofuscin; this
increase in concentrations of the toxic substance leads to the death of the
cell which in turn can lead to severe neurological impairment, with
examples including loss of vision, loss of control over body movements,
and seizures (Awano et al., 2006; Santavuori, 1988)
Nucleus:
The use of a haematoxylin stain is beneficial for the visualisation of the
nucleus as it stains basophilic structures blue (Tammen, 2016). Due to the
acidic nature of DNA this makes for the identification of nuclei quite
simplistic. The nuclei in Images C and D (Figure 1) can clearly be identified
as the blue oval-shaped structures. Nuclei are found in all eukaryotic cells
(except red blood cells) and are mostly round in shape however may differ
dependent upon the type of cell. The nucleus is the largest organelle of
the cell and is approximately 4-10 micrometres in diameter (Tammen,
2016). The nucleus is a double membrane bound organelle which is
responsible for the storage of genetic material in the form of chromatin.
Chromatin is a complex of DNA with associated histones. Chromatin is
found in two forms; heterochromatin which is inactive and stains densely
and euchromatin which is less densely packed (abcam, 2016). The nucleus
is separated from the cytoplasm by a bimembrane called the nuclear
envelope. This contains pores which are utilised for vesicle transport. The
outer membrane of the nuclear envelope is connected to the endoplasmic
reticulum which is the site of protein synthesis by bound ribosomes. DNA
obtained from the nucleus is transcribed by mRNA before being translated
by tRNA to form proteins (Tammen, 2016). The nucleolus is the region of
the nucleus in which ribosomal subunits are assembled. This assembly
begins with the transcription of pre-rRNA by the enzyme RNA polymerase I
(Olsen, 2015).

Lysosomes:
An organelle which is closely associated with Neuronal Ceroid
Lipofuscinosis (Battens Disease) is the lysosome. The disease is caused
by an abnormal build-up of the lipopigment lipofuscin inside the organelle
(lysosome) and promotes cell senescence (Jalanko & Braulke, 2009).
Unregulated cell death within neural areas is of particular concern as it
leads to neurological impairment. Lysosomes exhibit two main forms;
primary and secondary. The primary lysosomes are the resultant of a
budding from the Golgi apparatus. The secondary lysosomes are formed
when primary lysosomes bind to a vesicle and breaks down the material
encased in the vesicle. Lysosomes are encased by a phospholipid bilayer
which assists in containing the various digestive enzymes within (Ivy Rose,
2015). Lysosomes function to enzymatically digest material both up taken
from the external of the cell and expired cellular components within the
cell (BSCB, 2016). These enzymes which speed up degradation are
predominantly acid hydrolases (i.e. lipases, phosphatases) which function
at a slightly acidic pH of approx. 5 (Tammen, 2016). Lysosomes are
responsible for the degradation and recycling of a variety of cellular
materials. These come from 3 main sources; material taken into the cell
via endocytosis, material taken in via phagocytosis and thirdly, expired
material inside the cell taken in by autophagosomes. It has been
evidenced that the lysosome is not an independent organelle and requires
collaboration with late endosomes in order to digest material (BSCB,
2016). As aforementioned, lysosomes play an integrated role with other
cellular components in order to recycle and digest cellular material.
Lysosomes, or lysosomal complexes are often the final destination in
many secretory pathways following exocytosis. Their coordination with
late endosomes is essential for normal cellular function as the acid
hydrolases contained within the lysosome break down the material
provided by the endosome (Tammen, 2016).

References
Abitbol, M., 2010. A canine Arylsulfatase G (ARSG) mutation leading to a
sulftase deficiency is associated with neuronal ceroid lipofuscinosis.
Proceedings of the National Academy of Sciences of the United States of
America (PNAS), 17 August, 107(33), pp. 14775-14780.
Brunk, U. & Terman, A., 2002. Lipofuscin: mechanisms of age-related
accumulation and influence on cell function. Free Radical Biology and
Medicine, 1 September, 33(5), pp. 611-619.
Fischer, A., Jacobson, K. & Rose, J. Z. R., 2008. Hematoxylin and eosin
staining of tissue and cell sections. Cold Spring Harbor Protocols, 1 May, p.
4986.
Gilissen, E. & Staneva-Dobrovski, L., 2013. Distinct Types of Lipofusci
PIgment in the Hippocampus and Cerebellum of aged Cheirogaleid
Primates. The Anatomical Record, Issue 296, pp. 1895-1906.

Indiana, I. T. C. C. o., 2008. Week 11. [Online]

Available at:
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(CTSD) in American Bulldogs with neuronal ceroid lipofuscinosis.Molecular
genetics and metabolism, 87(4), pp.341-348.
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lipofuscinoses.Biochimica et Biophysica Acta (BBA)-Molecular Cell
Research, 1793(4), pp.697-709.
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and Development, 10(2), pp.80-83.