Developmental recapitulation of spinocerebellar ataxia

Spinocerebellar ataxias:
Patients with spinocerebellar ataxia (SCA) experience a gradual loss of
physical control while maintaining full mental capacity due to progressive
neurodegeneration of the cerebellum. This genetic disorder has 60 variants, of
which only 12 can be diagnosed with a blood test. As there is currently no cure
for SCAs, therapeutic rehabilitation of patients is used for alleviating some
of its symptoms such as, poor coordination of: gait, eye, speech and hand
movements. There are 29 gene mutations responsible for SCA which are classified
into autosomal dominant, autosomal recessive or X-linked. The SCA mutant genes
lead to neuron degeneration, specifically Purkinje neurons in almost all SCAs,
which undergo at least one of the following: iron stress (Anheim et al. 2012),
oxidative stress (Guo et al. 2010), mitochondrial dysfunction (Rugarli and
Langer 2012) or protein aggregation (Orr 2012). While the gene mutations of SCA
are categorized, the SCA pathology is still unclear with respect to whether
transcription regulation, import of proteins to nucleus, RNA splicing and
ubiquitination are causes or effects of SCA (Orr 2012). Mouse models cannot
completely reproduce the human disease because of their relatively short life
span as most SCAs in humans have an onset several decades into life. Another
factor to consider in mouse models is the expression of the mutant transgene in
all neurons vs. specific populations of neurons such as Purkinje neurons, which
more closely resembles the human disease. What is clear until now is that the
full complexity of the human disease has not been possible to be repeated in
mice (Ingram et al. 2012). Because neurons in culture have a shorter lifetime
than the lifetime of a mouse, we would use the cultures to identify the
molecular and electrophysiological events that precede the onset of Ataxia.
Thus, we do not expect to fully recapitulate the neurodegeneration in vitro, we
do however expect to replay some of the progression of the neuropathology.

From cerebellar development to spinocerebellar ataxia disease models:
While Purkinje cells of mouse models of SCA display alterations in innervations
(Barnes et al. 2011) and intrinsic firing rates (Shakkottai et al. 2011), it is
not clear which developmental stages lead to these phenotypes and which
molecular events precede them. In this study, we propose to recapitulate
cerebellar development to obtain adult cerebellar neurons through
differentiation of the induced pluripotent stem (IPS) cells of probands. Thus
far, human IPS cells have been differentiated into neurons for the purpose of
studying the molecular events that result in Ataxia telangectiasia (Carlessi et
al. 2013), which is the second most common autosomal recessive SCA.
Differentiation protocols of human IPS cells with or without the ataxia
telangiectasia gene have yielded GABAergic and glutamatergic (De Filippis et
al. 2007) and dopaminergic (Donato et al. 2007) neurons to study in vitro the
molecular onset of the disease (Carlessi et al. 2013). However, the current
challenge is to obtain specialized neurons from IPS cells; such as, Purkinje
neurons because these are particularly sensitive to mutant SCA genes. In this
regard, mouse embryonic stem cells have been successfully differentiated into
Purkinje neurons (Muguruma et al. 2010). Therefore, we would adapt this
protocol with embryonic stem cells (Muguruma et al. 2010) for the neuronal
differentiation of human IPS cells summarized in these three steps: IPS
culture, neuronal differentiation with fibroblast growth factor-2 (FGF2),
Insulin and cyclopamine, and adult neuron co-culture.

Because many ataxias are misdiagnosed or go years without knowing the exact
type, recapitulation of development of specialized neurons can yield a
molecular and electrophysiological profiling of healthy and diseased phenotypes
that is not possible to achieve in mouse models. For example, rapid
developments in microarray technologies would provide new opportunities to
correlate the molecular pathways stimulated in diseased neurons with their
electrophysiological phenotype.

1. We will recruit 6 probands who experience cerebellar movement disorders with
spinocerebellar ataxia features such as uncoordinated movement (i.e. asthenia,
asynergy, delayed reaction time and dyschronometria) in limbs and organs such
as the eyes. A skin biopsy and DNA from blood drawn would be used for the
generation of induced pluripotent stem (IPS) cells. It will be assessed if
cerebellar neurons can be obtained in vitro from the IPS cells of these
probands. If cerebellar neurons are produced, their biology will be evaluated
through immunohistochemistry with known markers of cerebellum physiology and
neurodegeneration and patch clamp techniques for electrophysiology.

2. We will recruit up to 5 first-, second-, or third degree biological
relatives of these probands who may or may not be affected with cerebellar
movement disorders. These subjects will also be clinically assessed with blood
drawn and a skin biopsy for the generation of cerebellar neurons. Cerebellar
development and neurodegeneration markers and electrophysiology of cerebellar
neurons will also be evaluated in these probands.

Definition of Subject Groups:

1. Probands (N = 6). These individuals who meet spinocerebellar ataxia
inclusion criteria for a cerebellar movement disorder will be fully clinically
assessed and have blood samples taken for the extraction of DNA and a skin
biopsy (4 mm).

2. Relatives of probands (N = 5). Available first-, second-, or third degree
biological relatives (both affected and unaffected) will be asked to contribute
a blood specimen for DNA testing. In addition, they will receive the same
clinical assessments as the probands. These family members will help elucidate
heritability patterns and to evaluate whether potentially pathological genetic
variants identified in this investigation co-segregate with the complex
phenotype in certain families.

Recruitment:
Probands: Recruitment is performed by a member of the research group and will
be done through referral by clinicians of the Erasmus MC department of
neuroscience. Referral can also be done by a family member or by confidential
screening of records with subsequent contact by a clinician. The investigator
(G. A. Higuera) will explain the study fully to the patient and provide an
information sheet to the patient. The patient will be able to read and retain
the details of the study (see informed consent sheet). After the patients have
time to consider the information fully and have been encouraged to ask
questions, they will be asked to give informed consent by signing and dating a
consent form. All consent forms should be signed and dated by the investigator.
Access to the patient notes for verification and auditing purposes will be
required and permission must be obtained as part of the consent process.
Consent forms will be reviewed at the trial center and retained in the
patient*s dossier by the investigator. A copy of the signed consent form will
be given to the patient. A separate log will include all patients (initials and
date of birth) screened for inclusion for the study. The principal
investigators will meet regularly with the clinicians to supervise recruitment
and selection of appropriate cases.

Family members: If the proband has signed his/her consent form, We will ask the
proband to explain to interested family members his or her own participation in
this study and give the family members the information letter and the research
team*s request to include them in the study. If this is not feasible, the
members of the research team will help wherever possible to track and contact
family members in order to explain to them the study, give them the information
letter and ask for their participation. Contacting family members of the
proband will be done only after having obtained permission from the proband.
Should the family members agree to participate after reading the information
letter, we will obtain their informed consent before receiving blood, clinical
or family history information.

Risks:
We expect no serious adverse events in this study. Peripheral venous blood
sampling (max. 30 mL) is a routine minimally-invasive procedure which will be
performed only by highly experienced and certified nurses and phlebotomists.
Further, our neuropsychological assessments are highly structured and have been
extensively tested, without any known serious adverse events.

Adverse events may include minor bruising or local tenderness at the site of
venous blood sampling. All patients will be monitored to ensure proper
hemostasis. During interviews and neuropsychological testing, the patient will
be fully aware of his/her right to terminate the testing at any time and for
any reason.

The Erasmus MC medical ethical committee has waived the obligation of insurance
for the participants in this study because it believes that this research has
little or no risk involved.

Compensation:
Probands and family members will be compensated for any travel costs incurred.
For the clinical assessment and blood sample, participants will be remunerated
with ¤50,- for their time and effort. For donating the skin biopsy,
participants will be remunerated with ¤50,- as well.